ATM kinase compositions and methods

ABSTRACT

The present invention provides methods for detecting activation of ATM kinase, DNA damage, and DNA damaging agents. Further provided are antibodies which specifically recognize the phosphorylation state of Ataxia Telangiectasia-Mutated (ATM) kinase. Methods of identifying agents which modulate the activation and activity of ATM kinase are also provided.

INTRODUCTION

[0001] This invention was made in the course of research sponsored by the National Institutes of Health (NIH Grant Nos. CA71387). The U.S. government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] Eukaryotic cells have evolved complex mechanisms to deal with environmental stresses. Signal transduction pathways are rapidly activated following exposure to DNA damaging agents and other cellular stresses, and these pathways affect processes such as gene transcription and cell cycle progression (Hartwell and Weinert (1989) Science 246:629-634; Hartwell and Kastan (1994) Science 266:1821-1828; Elledge (1996) Science 274:1664-1672). The protein encoded by the Ataxia-telangiectasia Mutated (ATM) locus, is a kinase critical for the initiation of signaling pathways following exposure of mammalian cells to ionizing radiation (IR) and to other agents that introduce double-strand breaks into cellular DNA (Kastan and Lim (2000) Mol. Cell Biol. 1:179-186; Shiloh and Kastan (2001) Adv. Cancer Res. 83:209-254). Cells from Ataxia-telangiectasia (A-T) patients typically lack detectable ATM protein, contain abnormalities in telomere morphology, and exhibit abnormal responses to IR, including increased cell death, increased chromosomal breakage, and cell cycle checkpoint defects (Shiloh (1997) Ann. Rev. Genet. 31:635-662). In addition, A-T patients exhibit progressive cerebellar ataxia, immune deficiencies, gonadal atrophy, oculocutaneous telangiectasias, radiation sensitivity, premature aging and increased risk of cancers, particularly lymphomas.

[0003] The ATM gene encodes a 370-kDa protein (Accession No. Q13315; SEQ ID NO: 1) that belongs to the phosphoinositide 3-kinase (PI-3K) superfamily (Savitsky, et al. (1995) Science 268:1749-1753) which phosphorylates proteins rather than lipids (Banin, et al. (1998) Science 281:1674-1677; Canman, et al. (1998) Science 281:1677-1679). The 350 amino acid kinase domain at the C-terminus of this protein is the only segment of ATM with an assigned function. Exposure of cells to IR triggers ATM kinase activity and this function is required for arrests in G1, S, and G2 phases of the cell cycle (Shiloh and Kastan (2001) Adv. Cancer Res. 83:209-254). Several substrates of the ATM kinase participate in these IR-induced cell cycle arrests. For example, phosphorylation of p53, mdm2, and Chk2 govern the G1 checkpoint (Banin, et al. (1998) Science 281:1674-1677; Canman, et al. (1998) Science 281:1677-1679; Maya, et al. (2001) Genes Dev. 15:1067-1077; Matsuoka, et al. (2000) Proc. Natl. Acad. Sci. USA 97:10389-10394; Chehab, et al. (2000) Genes Dev. 14:278-288); Nbs1, Brca1, FancD2, and SMC1 participate in the transient IR-induced S-phase arrest (Lim, et al. (2000) Nature 404:613-617; Wu, et al. (2000) Nature 405:477-482; Zhou, et al. (2000) J. Biol. Chem. 275:10342-10348; Taniguchi, et al. (2002) Cell 109:459-472; Kim, et al. (2002) Genes Dev. 16:560-570; Yazdi, et al. (2002) Genes Dev. 16:571-582; Xu, et al. (2002) Cancer Res. 62:4588-4591); and Brca1 and hRad17 have been implicated in the G2/M checkpoint (Xu, et al. (2001) Mol. Cell. Biol. 21:3445-3450; Bao, et al. (2001) Nature 411:969-974).

[0004] The mechanisms by which eukaryotic cells sense DNA strand breaks is unknown, but the rapid induction of ATM kinase activity following IR indicates that it acts at an early stage of signal transduction in mammalian cells (Banin, et al. (1998) Science 281:1674-1677; Canman, et al. (1998) Science 281:1677-1679). Transfected ATM is a phosphoprotein that incorporates more phosphate after IR treatment of cells (Lim, et al. (2000) Nature 404:613-617), suggesting that ATM kinase is itself activated by post-translational modification.

[0005] Inhibiting ATM for the treatment of neoplasms, particularly cancers associated with decreased p53 function, has been suggested (Morgan, et al. (1997) Mol. Cell Biol. 17:2020-2029; Hartwell and Kastan (1994) Science 266:1821-1828; Kastan (1995) New Eng. J. Med. 333:662-663; WO 98/56391). WO 98/56391 further provides genetically manipulated knock-out mice as a model for testing ATM inhibitors and suggests the use of an inhibitory antibody to ATM, a dominant-negative fragment of ATM or an ATM antisense strategy to inhibit ATM.

[0006] U.S. Pat. No. 6,387,640 discloses the use of an ATM kinase substrate recognition sequence in an assay system to screen for compounds that modulate ATM-mediated phosphorylation. The substrate recognition sequence provided comprises Xaa₁-Xaa-Xaa₂-Ser-Gln-Xaa-Xaa (SEQ ID NO: 2) wherein Xaa₁ is a hydrophobic amino acid, Xaa₂ is a hydrophobic amino acid or aspartic acid, and Xaa is any amino acid.

[0007] U.S. Pat. No. 6,348,311 further discloses a method of identifying an inhibitor of ATM-mediated kinase activity by determining the extent of cell survival after HTLV infection.

SUMMARY OF THE INVENTION

[0008] One aspect of the present invention provides a method of identifying the activation state of ATM kinase. In a cell, inactive ATM kinase is found as a homodimer or higher order multimer. Upon autophosphorylation of a serine corresponding to residue 1981 of ATM kinase (SEQ ID NO: 1), ATM becomes an active monomer. Accordingly, the method provides determining the phosphorylation state of serine 1981 (Ser¹⁹⁸¹) which is indicative of the activation state of ATM kinase. In a preferred embodiment, monoclonal or polyclonal antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ are used to determine the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase by immunoassay analysis.

[0009] Another aspect of the invention provides a method of detecting DNA damage in a sample comprising identifying the activation state of ATM kinase via the phosphorylation state of Ser¹⁹⁸¹. The method may be used to monitor the effectiveness of radiation therapy or chemotherapy.

[0010] Another aspect of the invention provides a method of detecting DNA damaging agents in a biological or environmental sample comprising identifying the activation state of ATM kinase via the phosphorylation state of Ser¹⁹⁸¹. A kit for detecting a DNA damaging agent is also provided.

[0011] A further aspect of the invention provides a method of producing soluble ATM kinase by contacting a first polypeptide of ATM kinase containing the kinase domain with a second polypeptide of ATM kinase containing Ser¹⁹⁸¹. The first and second polypeptides may be produced separately or as a single polypeptide in the same cell.

[0012] A further aspect of the invention provides a cell-based assay for identifying agents which modulate the activation of ATM kinase. The method provides contacting cells containing ATM kinase with an agent and determining whether said agent agonizes or antagonizes the activation of ATM kinase in the cell. The activation state of ATM kinase is identified via the phosphorylation state of Ser¹⁹⁸¹.

[0013] A further aspect of the invention provides a cell-free assay for identifying agents which modulate ATM kinase activity. The method provides contacting soluble ATM kinase protein with an agent and ATP and determining whether said agent agonizes or antagonizes the ATM kinase activity.

[0014] These and other aspects of the present invention are set forth in more detail in the following description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] It has now been found that the major site of ATM kinase phosphorylation is located at serine residue 1981 (Ser¹⁹⁸¹) of ATM kinase (SEQ ID NO: 1), also referred to herein as ATM. Phosphorylation of Ser¹⁹⁸¹ results from intermolecular autophosphorylation. The phosphorylation of Ser¹⁹⁸¹ does not directly regulate the activity of the kinase, but instead disrupts ATM kinase oligomers which in turn allows accessibility of substrates to the ATM kinase domain. The rapidity and stoichiometry of the phosphorylation reaction indicate that ATM is not activated by binding directly to DNA strand breaks. While not wishing to be bound by any particular theory, it is believed that DNA damage rapidly causes changes in higher order chromatin structures that initiate this activation.

[0016] Not to be held to any one particular mechanism of action, it is believed that in the unperturbed cell, ATM is sequestered as a dimer or higher order multimer with its kinase domain bound to an internal domain of a neighboring ATM molecule containing Ser¹⁹⁸¹. In this complex, ATM is unable to phosphorylate other cellular substrates. Following DNA damage, the kinase domain of one ATM molecule phosphorylates Ser¹⁹⁸¹ of an interacting ATM molecule, and the phosphorylated ATM is then dissociated from the complex and is freed to phosphorylate other substrates in the cell. The kinase dead and non-phosphorylatable mutants of ATM retain endogenous ATM in a complex since they cannot be phosphorylated and released after IR, thus inhibiting cellular ATM activity. This mechanism provides an explanation for the dominant inhibitory property of kinase-inactive ATM.

[0017] Several different molecular mechanisms have been identified that regulate the activity of protein kinases. Protein kinases are generally restrained in an inactive state with the acquisition of catalytic activity controlled at multiple levels, ranging from the binding of allosteric factors to changes in the subcellular localization of the enzyme (Huse and Kuriyan (2002) Cell 109:275-282). Since all protein kinases catalyze the same reaction, their active conformations tend to be structurally similar. However, different classes of kinases have evolved distinct inactive states and adoption of the catalytic conformation of the enzyme can be impeded in different ways. These include steric hindrance of substrate access to the catalytic domain by an activation loop that is often controlled by phosphorylation (Johnson and Noble (1996) Cell 85:149-158); allosteric regulation of the activation loop via, for example, the PSTAIRE helix in the cyclin dependent kinase family (De Bondt, et al. (1993) Nature 363:595-602; pseudosubstrate inhibition of both substrate and nucleotide binding as seen in twitchin (Hu, et al. (1994) Nature 369:581-584); and intramolecular autoinhibition by N-terminal segments that inhibit catalytic activity, as in the case of the EphB2 receptor kinase (Dodelet, and Pasquale (2000) Oncogene 19:5614-5619). The results provided herein indicate a novel mechanism of kinase activation in which the cellular activity of one ATM kinase molecule is impeded by intermolecular association with an internal domain of a partner ATM molecule; access of substrates to the catalytic domain is impeded by this association. This type of regulation is similar to pseudosubstrate inhibition, with the major variations being that the pseudosubstrate is a domain of itself (albeit in trans) and that this partner is not a mimic, but actually becomes a substrate in order to release the inhibition.

[0018] Accordingly, the present invention provides compositions and methods for identifying the activation state of ATM kinase by determining the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase. The activation of ATM kinase is indicative of DNA damage; thus, the invention further provides methods for detecting DNA damage and DNA damaging agents. Further provided is a method for producing soluble ATM kinase by combining a first polypeptide of ATM kinase containing the kinase domain with a second polypeptide of ATM kinase containing Ser¹⁹⁸¹. Methods of identifying agents which modulate activation of ATM kinase in a cell and ATM kinase activity are also provided.

[0019] One aspect of the invention provides a method of identifying the activation state of ATM kinase in a cell. In an unperturbed cell (i.e., a cell in which no DNA damage has occurred), ATM kinase is found in a dimer or multimer which is the inactivate state wherein ATM is unable to phosphorylate other cellular substrates. Upon DNA damage, ATM autophosphorylates Ser¹⁹⁸¹ and is converted into a monomer which is the active state wherein ATM is able to phosphorylate other cellular substrates. Therefore, in a cell, the active and inactive states (i.e., activation states) of ATM kinase are distinguishable by the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase (SEQ ID NO: 1). As one of skill in the art can appreciate, the activation state of any homolog or mutant of ATM kinase may be identified by determining the phosphorylation state of a serine corresponding to residue 1981 of ATM kinase. The location of the critical serine residue corresponding to Ser¹⁹⁸¹ of ATM kinase can readily be determined by comparing the sequence of ATM kinase (SEQ ID NO: 1) with the sequence of homologs or mutants of ATM kinase.

[0020] In general, the method of identifying the activation state of ATM kinase comprises obtaining a sample such as a biopsy sample, tissue, cell or fluid (e.g., whole blood or plasma) isolated from a subject and determining the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase in the sample. Kinase inhibitors may be present during the isolation of ATM kinase to preserve the phosphorylation state of Ser¹⁹⁸¹ as it would have been found in the cell prior to the isolation step. It is contemplated that the phosphorylation state of Ser¹⁹⁸¹ of ATM may be determined using a variety of separation and/or detection methods, including those exemplified herein. For example, [³²P] phosphorylated ATM is digested with trypsin and separated by well-known conventional column chromatography, 2-D gel electrophoresis, or capillary electrophoresis methodologies. For separation by column chromatography, reverse-phase HPLC may be employed with collection via peak detection. Under the conditions used for reverse-phase HPLC (0.05% TFA, pH 2.2), a phosphorylated peptide generally elutes slightly earlier than the corresponding non-phosphorylated peptide and may or may not be separated from it. Once HPLC fractions containing the Ser¹⁹⁸¹ phosphorylated peptide are located by Cerenkov counting, a small aliquot of each may be analyzed by MALDI-MS.

[0021] As an alternative to radiolabelling, western blots made from 2-D gels may be probed using anti-phosphoserine antibodies (Research Diagnostics, Inc., Flanders, N.J.) to recognize the degree of phosphorylation of a peptide fragment containing Ser¹⁹⁸¹.

[0022] Alternatively, one may use a phosphoprotein purification kit (QIAGEN®, Valencia, Calif.) for separation of the phosphorylated from the unphosphorylated cellular protein fraction. The affinity chromatography procedure, in which phosphorylated proteins are bound to a column while unphosphorylated proteins are recovered in the flow-through fraction, reduces complexity and greatly facilitates phosphorylation-profile studies. ATM may then be purified from each fraction and the degree of phosphorylation of a peptide fragment containing ser¹⁹⁸¹ determined via autoradiography or immunoassays.

[0023] In a preferred embodiment, the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase is detected using antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase (SEQ ID NO: 1). Such antibodies may be utilized with or without purification, fragmentation, or fractionation or ATM. An antibody which specifically recognizes the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase (SEQ ID NO: 1) comprises polyclonal antibody α-Ser¹⁹⁸¹ and monoclonal antibodies 7C10, 12E10, 13C5, 13H4, 2H12, 7A4, 9D8 and 10H11, which specifically recognize unphosphorylated Ser¹⁹⁸¹; and polyclonal antibody α-Ser¹⁹⁸¹-P, which specifically recognize phosphorylated Ser¹⁹⁸¹. An antibody is said to specifically recognize the phosphorylation state of Ser¹⁹⁸¹ if it is able to discriminate between the unphosphorylated and phosphorylated forms of Ser¹⁹⁸¹ and bind ATM to form an ATM kinase-antibody complex, i.e., antigen-antibody complex. For example, an antibody which specifically recognizes the unphosphorylated state of Ser¹⁹⁸¹ will only bind to an ATM kinase with an unphosphorylated Ser¹⁹⁸¹ and not to an ATM kinase with a phosphorylated Ser¹⁹⁸¹ (e.g., α-Ser¹⁹⁸¹). Likewise, an antibody which specifically recognizes the phosphorylated state of Ser¹⁹⁸¹ will only bind to an ATM kinase with a phosphorylated Ser¹⁹⁸¹ and not to an ATM kinase with an unphosphorylated Ser¹⁹⁸¹ (e.g., α-Ser¹⁹⁸¹-P, 7C10, 12E10, 13C5, 13H4, 2H12, 7A4, 9D8, 10H11).

[0024] In general, a method of using antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ in the identification of the activation state of ATM kinase provides contacting a sample with said antibody and detecting the formation of an antigen-antibody complex using an immunoassay. The ATM kinase antigen, as used herein, includes both the phosphorylated and unphosphorylated states of Ser¹⁹⁸¹, however, the phosphorylated state is preferred. The conditions and time required to form the antigen-antibody complex may vary and are dependent on the sample being tested and the method of detection being used. Once non-specific interactions are removed by, for example, washing the sample, the antigen-antibody complex is detected using any one of the well-known immunoassays used to detect and/or quantitate antigens. Exemplary immunoassays which may be used in the methods of the invention include, but are not limited to, enzyme-linked immunosorbent, immunodiffusion, chemiluminescent, immunofluorescent, immunohistochemical, radioimmunoassay, agglutination, complement fixation, immunoelectrophoresis, western blots, mass spectrometry, antibody array, and immunoprecipitation assays and the like which may be performed in vitro, in vivo or in situ. Such standard techniques are well-known to those of skill in the art (see, e.g., “Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al., “Methods and Immunology”, W. A. Benjamin, Inc., 1964; and Oellerich, M. (1984) J. Clin. Chem. Clin. Biochem. 22:895-904; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988) 555-612).

[0025] These immunoassays typically rely on labeled antigens, antibodies, or secondary reagents for detection. These proteins may be labeled with radioactive compounds, enzymes, biotin, or fluorochromes. Of these, radioactive labeling may be used for almost all types of assays. Enzyme-conjugated labels are particularly useful when radioactivity must be avoided or when quick results are needed. Biotin-coupled reagents usually are detected with labeled streptavidin. Streptavidin binds tightly and quickly to biotin and may be labeled with radioisotopes or enzymes. Fluorochromes, although requiring expensive equipment for their use, provide a very sensitive method of detection. Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof may be accomplished using standard techniques (see, for example, Kennedy, et al. (1976) Clin. Chim. Acta 70:1-31 and Schurs, et al. (1977) Clin. Chim Acta 81:1-40).

[0026] In accordance with identifying the activation state of ATM kinase, the presence or absence of the antigen-antibody complex is correlated with active or inactive ATM kinase in a sample, respectively. For example, a sample to which α-Ser¹⁹⁸¹-P binds is indicative of the presence of active ATM kinase in said sample.

[0027] As provided herein, monoclonal and rabbit polyclonal antibodies that specifically recognize Ser¹⁹⁸¹, only when it is in either the unphosphorylated (α-Ser¹⁹⁸¹) or phosphorylated (α-Ser¹⁹⁸¹-P, 7C10, 12E10, 13C5, 13H4, 2H12, 7A4, 9D8, 10H11) state were generated. Initial specificity of these antibodies for appropriate peptides was demonstrated by ELISA and by specific recognition and blocking on dot blots. The specificity of the antibodies were confirmed on western blots of immunoprecipitated FLAG®-tagged ATM protein where wild-type and kinase-inactive ATM were recognized by both antisera, but ATM protein with Ser¹⁹⁸¹ mutated to alanine (Ser¹⁹⁸¹→Ala) was not recognized by either antisera. The relative amount of wild-type ATM recognized by the α-Ser¹⁹⁸¹ antisera was reduced several-fold within 30 minutes after exposure of the cells to 10 Gy IR, whereas the relative amount of kinase-inactive ATM that was recognized remained the same. Conversely, the relative amount of wild-type ATM recognized by the α-Ser -¹⁹⁸¹-P antisera was increased several-fold 30 minutes after treatment with 10 Gy IR while recognition of kinase-inactive ATM was unaffected. These results mirror the metabolic labeling results using transfected wild-type ATM and kinase-inactive ATM in 293T cells described above.

[0028] Upon exposure to IR and UV, endogenous ATM is phosphorylated at Ser¹⁹⁸¹. Non-transformed, exponentially growing primary human fibroblasts were exposed to either 10 Gy IR or 10 J/m² UV. The α-Ser¹⁹⁸¹-P antisera did not bind to ATM protein immunoprecipitated from unirradiated cells, but recognized ATM one hour following exposure to IR and five hours following exposure to both IR and UV. The relative amount of Ser¹⁹⁸¹ phosphorylation seen five hours following IR treatment was several-fold higher than that seen following UV. This differential recognition was not due to changes in cell cycle distribution which did not change significantly in the first hour after IR. Moreover, primary fibroblasts arrested in G₀ also demonstrate this phosphorylation event following exposure to either IR or UV irradiation.

[0029] Antibodies provided in the present disclosure are of the monoclonal and polyclonal type. It is contemplated that such antibodies may be natural or partially or wholly synthetically produced. All fragments or derivatives thereof which maintain the ability to specifically bind to and recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase are also contemplated. The antibodies may be a member of any immunoglobulin class, including any of the classes: IgG, IgM, IgA, IgD, and IgE. Derivatives of the IgG class, however, are preferred in the present invention.

[0030] ATM kinase antibody fragments may be any derivative of an antibody which is less than full-length. Preferably, the antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, scFv, Fv, dsFv diabody, or Fd fragments. The antibody fragment may be produced by any means. For instance, the antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or it may be recombinantly produced from a gene encoding the partial antibody sequence. The antibody fragment may optionally be a single-chain antibody fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for instance, by disulfide linkages. The fragment may also optionally be a multi-molecular complex. A functional antibody fragment will typically comprise at least about 50 amino acids and more typically will comprise at least about 200 amino acids. As used herein, an antibody also includes bispecific and chimeric antibodies.

[0031] Naturally produced antibodies may be generated using well-known methods (see, e.g., Kohler and Milstein (1975) Nature 256:495-497; Harlow and Lane, In: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)). Alternatively, ATM kinase antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase are derived by a phage display method. Methods of producing phage display antibodies are well-known in the art (e.g., Huse, et al. (1989) Science 246(4935):1275-81).

[0032] Selection of ATM kinase-specific antibodies is based on binding affinity to ATM kinase which is either phosphorylated or unphosphorylated at Ser¹⁹⁸¹ and may be determined by the various well-known immunoassays indicated above.

[0033] Another aspect of the present invention provides a method of detecting DNA damage in a cell by identifying the activation state of ATM kinase. It has now been shown that DNA damaging agents which introduce breaks in the phosphodiester backbone of DNA induce rapid, detectable phosphorylation of Ser¹⁹⁸¹ and hence activation of ATM kinase. Ser¹⁹⁸¹ phosphorylation was examined in exponentially growing primary fibroblasts one and five hours after treatment with IR, thymidine block, the ribonucleotide reductase inhibitor hydroxyurea (HU), the topoisomerase inhibitors camptothecin and etoposide, the DNA alkylating agent methlymethanesulfonate (MMS), the DNA polymerase α inhibitor aphidicolin, or the oxidizing agent H₂O₂. Phosphorylation of Ser¹⁹⁸¹ in ATM was detected one hour following exposure to 10 Gy IR, 2 nM camptothecin, 17 nM etoposide or 0.1% H₂O₂, all of which are DNA damaging agents. The only significant change in this pattern five hours after each of these treatments was more prominent phosphorylation following exposure to the alkylating agent, MMS. The delayed Ser¹⁹⁸¹ phosphorylation after MMS was similar to that observed following UV irradiation, an agent which, like MMS, damages DNA bases, but can induce DNA strand breaks either through repair processes or by DNA replication past DNA adducts. Little or no phosphorylation of ATM was apparent over this time frame following exposure to the DNA synthesis inhibitors hydroxyurea, thymidine, or aphidicolin, none of which directly damage DNA. Thus, all agents tested that directly damage DNA by, for example, inducing DNA strand breaks, induced rapid phosphorylation of Ser¹⁹⁸¹, whereas treatments that primarily inhibit DNA synthesis failed to do so.

[0034] The kinetics, dose responsiveness and stoichiometry of ATM autophosphorylation following IR were also examined in primary fibroblasts. Over a 24-hour time frame, phosphorylation of Ser¹⁹⁸¹ was maximal by 15 minutes after exposure to 2 Gy IR and remained stable and detectable for at least 24 hours thereafter. Moreover, phosphorylation of Ser¹⁹⁸¹ was detected immediately upon cellular harvesting following the 30 second exposure required to deliver 0.5 Gy IR and was maximal by five minutes. Initial dose responsiveness was evaluated over a range from 1 to 9 Gy, but induction was already maximal at 1 Gy at the 30 minute time point used. A more detailed evaluation of doses less than 1 Gy provided that phosphorylation of Ser¹⁹⁸¹ was detectable following 0.11 Gy and was already maximal following 0.44 Gy at a 15 minute time point.

[0035] As the phosphorylation of Ser¹⁹⁸¹ in ATM can be detected following exposure to doses of IR as low as 0.11 Gy, which theoretically should cause just four double-strand breaks in the genomic DNA of a human diploid cell (Rogakou, et al. (1998) J. Biol. Chem. 273:5858-5868), the minimal number of DNA double-strand breaks that would induce detectable ATM phosphorylation was determined. SV-40-transformed fibroblasts were obtained that had been stably transfected with a plasmid containing a sequence that can be cut by the restriction enzyme I-SceI, a site which has not been found in any mammalian genome (Richardson, et al. (1999) Methods Mol. Biol. 113:453-463). Southern blotting demonstrated that the genome of this cell line contained two copies of the I-SceI site. The (α-Ser¹⁹⁸¹-P antibody was able to detect Ser¹⁹⁸¹ phosphorylation of FLAG®-tagged, wild-type ATM that was co-transfected with I-SceI. No phosphorylation was seen in control transfectants where no active I-SceI was introduced or when either kinase-inactive ATM or ATM mutated at Ser¹⁹⁸¹ was utilized. Therefore, the α-Ser¹⁹⁸¹-P antibody can detect the introduction of as few as two DNA double-strand breaks in cells.

[0036] To estimate the fraction of cellular ATM protein that becomes phosphorylated after DNA damage, sequential immunoprecipitations of ATM from irradiated primary fibroblasts were performed with a conventional anti-ATM antibody and with the α-Ser¹⁹⁸¹-P antisera. In the absence of insult, the conventional anti-ATM antibody was able to immunoprecipitate virtually all of the ATM in the first absorption from unirradiated cells, while the α-Ser¹⁹⁸¹-P antisera brought down almost no ATM. The little ATM that was immunoprecipitated by the α-Ser¹⁹⁸¹-P antisera was not recognized by this antibody on western blots, likely due to a very small amount of non-phospho-specific antisera in the polyclonal antibody preparation. Following exposure to 0.5 Gy IR, the amount of ATM immunoprecipitated by the α-Ser¹⁹⁸¹-P antisera in the first absorption was similar to the amount of ATM immunoprecipitated by the conventional anti-ATM antibody and was greater than the remaining cellular ATM that was brought down in the second absorption by the conventional anti-ATM antibody. Since the conventional anti-ATM antibody was not completely efficient in bringing down all of the cellular ATM with a single immunoprecipitation from the irradiated cells it was estimated that at least 50% of the total ATM in an exponentially growing culture of primary human fibroblasts is autophosphorylated on Ser¹⁹⁸¹ by 15 minutes after exposure to 0.5 Gy IR.

[0037] Since such a high fraction of cellular ATM becomes phosphorylated so rapidly in the presence of so few DNA strand breaks, it is unlikely that the ATM oligomers could require direct binding to DNA strand breaks for activation and autophosphorylation. Therefore, the introduction of DNA strand breaks may cause a change in the nucleus that activates ATM at a distance from the break itself. As DNA strand breaks introduced by ionizing irradiation rapidly alter topological constraints on DNA (Roti Roti and Wright (1987) Cytometry 8:461-467; Jaberaboansari, et al. (1988) Radiat. Res. 114:94-104; Malyapa, et al. (1996) Int. J. Radiat. Oncol. Biol. Phys. 35:963-973), alterations in some aspect of chromatin structure fit the criteria of being a rapid change and being able to act a distance in the nucleus. Chromatin and chromosome structures can be altered by hypotonic conditions (Earnshaw, and Laemmli (1983) J. Cell Biol. 96:84-93; Jeppesen, et al. (1992) Chromosoma 101:322-332), therefore ATM phosphorylation following brief exposures of cells to mildly hypotonic buffers was examined. This minimally invasive treatment of cells induced rapid and diffuse phosphorylation of ATM protein as assessed by immunoblot and immunofluorescence. No phosphorylation of histone H2AXγ was observed, therefore there was no evidence for the introduction of DNA strand breaks with this treatment. In contrast, phosphorylation of both ATM and H2AXγ were apparent following IR. The patterns of immunofluorescent staining for ATM following that at the earliest time points, the staining was diffuse across the nucleus, but after several minutes, some foci were seen in addition to the diffuse nuclear staining. This pattern is consistent with a diffuse activation of ATM and migration of a fraction of ATM protein to the sites of DNA strand breaks to phosphorylate substrates at the breaks.

[0038] Accordingly, in another preferred embodiment DNA damage in a sample is detected by identifying the activation state of ATM kinase. The method provides obtaining a sample from a subject and determining the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase. The phosphorylation state of Ser¹⁹⁸¹ of ATM kinase may be determined using any one of the techniques provided herein, however, it is preferred that antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase be used. A sample containing a phosphorylated Ser¹⁹⁸¹ of ATM kinase, as determined by, for example, the binding of α-Ser¹⁹⁸¹-P antibody, is indicative of active ATM kinase and hence DNA damage in the subject from which the sample was obtained. Accordingly, a method of detecting DNA damage may be used as part of a screen in subjects suspected of having been exposed to a DNA damaging agent. Moreover, the detection method of the invention may be used alone or in combination with other well-known diagnostic methods to confirm DNA damage.

[0039] Damage to DNA may have a genetic- or age-related basis or may result from exposure to agents including those which generate DNA adducts by alkylation (e.g., methylmethane sulfonate (MMS), ethylmethane sulfonate (EMS), N-methyl-N-nitro-N-nitrosoguanine (MNNG), dimethylnitrosamine (DMN), dimethyl sulfate), and form intra- and inter-strand crosslinks (e.g., mitomycin C, psoralens). Furthermore, exposure to base analogs, such as bromouracil and aminopurine; nitrous acid; large molecules which bind to bases in DNA and cause them to be noncoding, i.e., “bulky” lesions; chemicals causing DNA strand breaks (e.g., peroxides); and radiation such as ultraviolet and ionizing radiation (e.g., X- and gamma-rays) also result in DNA damage.

[0040] Detection of DNA damage in a cell as determined by the activation state of ATM kinase is also useful for monitoring therapeutic effects during clinical trials and other treatment. Thus, the therapeutic effectiveness of an agent, such as a radionucleide for radiation therapy or a cytotoxic agent for chemotherapy designed to cause DNA damage in a cell, can be monitored using the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase, i.e., active ATM kinase, as an end-point target.

[0041] A further aspect of the invention provides a method of detecting a DNA damaging agent in a sample. A sample may be either of biological or environmental origin. Biological samples include those provided above as well as food products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Environmental samples include environmental material such as surface matter, soil, water, wastewater, sewage, sludge, industrial samples (e.g., industrial water), as well as samples obtained from food and dairy processing instruments, apparatus, equipment, disposable and non-disposable items. In addition to these environmental samples, it is contemplated that drinking water may be used with the method of the present invention. It is intended that the term drinking water encompass all types of water used for consumption by humans and other animals, including but not limited to well water, run-off water, water stored in reservoirs, rivers, streams, etc. The method provides contacting a test cell, which can be of prokaryotic or eukaryotic origin, containing ATM kinase, with a sample suspected of having a DNA damaging agent, allowing the test cell to incubate in the presence of the sample, and detecting whether DNA damage has occurred in the test cell by identifying the activation state of ATM kinase in said cell. Methods for identifying the activation state of ATM kinase in a cell are provided herein. In a preferred embodiment, active ATM kinase, in a cell exposed to a DNA damaging agent, is identified by determining the phosphorylation state of Ser¹⁹⁸¹ using antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase.

[0042] A further aspect of the invention provides a kit to detect a DNA damaging agent in a sample. The kit comprises antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase and are preferably labeled. The kit may further comprise a test cell containing ATM kinase. Further provided in the kit may be a means for determining the antigen-antibody complex using, for example, an immunoassay, and means for comparing the amount of antigen-antibody complex with a standard. The kit may be packaged in a suitable container and further comprise instructions for using the kit to detect DNA damaging agents.

[0043] A further aspect of the invention provides a method of producing soluble ATM kinase by contacting the kinase domain of ATM with a fragment of ATM kinase containing Ser¹⁹⁸¹. The domain of ATM containing Ser¹⁹⁸¹ was found to stably interact with the kinase domain of ATM. A GST fusion protein containing residues 1961-2046 of ATM (GST-ATM¹⁹⁶¹⁻²⁰⁴⁶) was co-expressed in E. coli with a 6×-Histidine (6×-His) fusion protein containing the C-terminal kinase domain of ATM, residues 2712-3056. Although the kinase domain was insoluble in bacteria when co-transfected with GST alone or a GST fusion with the ATM target peptide p53 (residues 1-101), a significant percentage was stabilized and solubilized in the presence of the GST-ATM¹⁹⁶¹⁻²⁰⁴⁶ fusion protein. The kinase domain was also solubilized in the presence of the GST-ATM¹⁹⁶¹⁻²⁰⁴⁶ fusion protein containing Ser¹⁹⁸¹→Ala, but remained insoluble in the presence of phosphorylation-mimic fusion peptides, Ser¹⁹⁸¹→Asp and Ser¹⁹⁸¹→Glu. Furthermore, the soluble kinase domain co-purified on glutathione-agarose with the GST-ATM¹⁹⁶¹⁻²⁰⁴⁶ fusion proteins containing wild-type sequence or Ser¹⁹⁸¹→Ala. These results indicate that the kinase domain and phosphorylation domain can stably bind to one another and that sequences flanking Ser¹⁹⁸¹ are critical for this interaction. The interaction was prevented by mutation of Ser¹⁹⁸¹ to either aspartic acid (Asp) or glutamic acid (Glu), both of which have charged side chains that mimic serine phosphorylation, indicating that phosphorylation of Ser¹⁹⁸¹ may prevent interaction of this domain with the kinase domain.

[0044] Diploid human fibroblasts were exposed to a range of formaldehyde concentrations to covalently crosslink endogenous ATM into a minimal complex. A prominent complex containing ATM was immunoprecipitated from cells following treatment of cells with 0.5 mM or 1.0 mM formaldehyde for 10 minutes. This complex migrated electrophoretically considerably slower than the denatured ATM monomer, which runs at 370-kDa, and beyond the range of conventional molecular weight markers. Furthermore, this ATM-containing complex disappeared after exposure of the cells to 10 Gy IR.

[0045] The number of ATM molecules present in the complex as well as the dependence of Ser¹⁹⁸¹ phosphorylation on the dissociation of the complex was determined. Hemagglutinin-tagged (HA-tagged) ATM was transfected into 293T cells along with wild-type, kinase-inactive, or Ser¹⁹⁸¹→Ala FLAG®-tagged ATM. In the absence of formaldehyde crosslinking, HA-tagged ATM was immunoprecipitated by anti-FLAG® Sepharose in association with each of the three FLAG®-tagged ATM proteins. Following 2 Gy IR, HA-tagged ATM was immunoprecipitated with both kinase-inactive and Ser¹⁹⁸¹→Ala FLAG®-tagged ATM, but was no longer bound to wild-type FLAG®-tagged ATM. In the absence of irradiation, the relative amounts of kinase-inactive and Ser¹⁹⁸¹→Ala FLAG®-tagged ATM that co-immunoprecipitated with HA-tagged ATM were lower than that of wild-type FLAG®-tagged ATM. This indicated that the association of wild-type, FLAG®-tagged ATM with wild-type, HA-tagged ATM was more stable than that of the mutant, FLAG®-tagged ATMs with HA-tagged ATM. Therefore, ATM exists as a dimer or higher order multimer in unperturbed cells and that intermolecular ATM autophosphorylation on Ser¹⁹⁸¹ is required for the dissociation of the complex following DNA damage.

[0046] As the ATM kinase domain interacts stably with the domain containing the autophosphorylation site, it was determined whether a truncated recombinant ATM molecule that included the phosphorylation and kinase domains would fold properly and have kinase activity. A galactose-inducible pYES plasmid containing 6×-His-FLAG®-tagged ATM¹⁹²³⁻³⁰⁵⁶ was transfected into Saccharomyces cerevisiae and a soluble ATM fragment was recovered. The polypeptide was purified by anti-FLAG® affinity chromatography. Following elution with synthetic FLAG® peptide and subsequent size-exclusion chromatography, the highly purified ATM fragment had kinase activity, exhibited wortmannin-sensitive phosphorylation of a p53 target peptide and autophosphorylation of Ser¹⁹⁸¹. Some of this ATM fragment migrated at the size of a dimer even under the harsh denaturing conditions of the SDS-PAGE gel indicating that the ATM protein homodimerizes in cells.

[0047] Accordingly, in another embodiment of the invention, soluble ATM kinase is produced by contacting a first polypeptide comprising the kinase domain of ATM with a second polypeptide comprising a fragment of ATM kinase containing Ser¹⁹⁸¹. The first and second polypeptide may be produced separately or produced as a single polypeptide. When produced separately, the first polypeptide containing the kinase domain may comprise the entire ATM kinase (i.e., full-length ATM kinase; residues 1-3056 of SEQ ID NO: 1) or a kinase domain fragment which retains the activity of ATM kinase (e.g., residues 2712-3056 of SEQ ID NO: 1). Furthermore, the second polypeptide containing Ser¹⁹⁸¹ may comprise a polypeptide of 50, 75, 85, or 95 amino acid residues with Ser¹⁹⁸¹ located approximately in the center of the polypeptide. An exemplary second polypeptide comprises residues 1961-2046 of SEQ ID NO: 1. When produced as a single polypeptide, the nucleic acid sequences encoding the first and second polypeptide reside on a single contiguous nucleic acid molecule, i.e., translated from one messenger RNA. A single polypeptide may comprise, for example, residues 1923-3056 or 1961-3056 of SEQ ID NO: 1. Alternatively, the single polypeptide may have the first and second polypeptide separated by a linker peptide ranging from 1 to 1000 amino acid residues (e.g., Gly-Ser-Gly, (Gly)₃, and the like).

[0048] When produced separately, the first and second polypeptide may be expressed from the same or separate expression vectors, from the same or different promoters, in the same or separate cells and combined for purification. Preferably the first and second polypeptide are expressed in the same cell.

[0049] Whether produced as a single polypeptide or as separate first and second polypeptides, the soluble ATM kinase will be referred to hereinafter as the ATM kinase polypeptide or simply ATM kinase in the context of producing soluble ATM kinase. Methods of producing ATM kinase in vivo (i.e., cell-based) are provided, however, as will be appreciated by one of skill in the art, ATM kinase may be produced using well-known in vitro transcription and translation methods.

[0050] Nucleic acids encoding ATM kinase may be incorporated into a recombinant expression vector in a form suitable for expression of the proteins in a host cell. A suitable form for expression provides that the recombinant expression vector includes one or more regulatory sequences operatively-linked to the nucleic acids encoding ATM kinase in a manner which allows for transcription of the nucleic acids into mRNA and translation of the mRNA into the protein. Regulatory sequences may include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are known to those skilled in the art and are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transfected and/or the level of expression required.

[0051] The soluble ATM kinase of the invention may be expressed not only directly, but also as a fusion protein with a heterologous polypeptide, i.e. a signal sequence for secretion and/or other polypeptide which will aid in the purification of ATM kinase. Preferably, the heterologous polypeptide has a specific cleavage site to remove the heterologous polypeptide from ATM kinase.

[0052] In general, a signal sequence may be a component of the vector and should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For production in a prokaryote, a prokaryotic signal sequence from, for example, alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders may be used. For yeast secretion, one may use, e.g., the yeast invertase, alpha factor, or acid phosphatase leaders, the Candida albicans glucoamylase leader (EP 362,179), or the like (see, for example WO 90/13646). In mammalian cell expression, signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex glycoprotein D signal may be used.

[0053] Other useful heterologous polypeptides which may be fused to ATM kinase include those which increase expression or solubility of the fusion protein or aid in the purification of the fusion protein by acting as a ligand in affinity purification. Typical fusion expression vectors include those exemplified herein as well as pGEX (Amersham Pharmacia Biotech, Uppsala, Sweden; Smith, and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse GST, maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0054] Eukaryotic microbes such as yeast may be transformed with suitable vectors containing nucleic acids encoding ATM kinase. Saccharomyces cerevisiae is the most commonly studied lower eukaryotic host microorganism, although a number of other species are commonly available. Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, nucleic acid sequences encoding ATM kinase, sequences for polyadenylation and transcription termination, and nucleic acid sequences encoding a selectable marker. Exemplary plasmids include YRp7 (Stinchcomb, et al. (1979) Nature 282:39; Kingsman, et al. (1979) Gene 7:141; Tschemper, et al. (1980) Gene 10:157), pYepSec1 (Baldari, et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz, et al. (1987) Gene 54:113-123), and pYES2 (INVITROGEN™ Corporation, San Diego, Calif.). These plasmids contain genes such as trp1, which provides a selectable marker for a mutant strain of yeast lacking the ability to grow in the presence of tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics 85:12). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0055] Suitable sequences for promoting ATM kinase expression in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman, et al. (1980) J. Biol. Chem. 255:2073) or other glycolytic enzymes (Hess, et al. (1968) J. Adv. Enzyme Reg. 7:149; Holland, et al. (1978) Biochemistry 17:4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further disclosed in EP 73,657.

[0056] In plant cells, expression systems are often derived from recombinant Ti and Ri plasmid vector systems. In the cointegrate class of shuttle vectors, the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation. Exemplary vectors include the pMLJ1 shuttle vector (DeBlock, et al. (1984) EMBO J. 3:1681-1689) and the non-oncogenic Ti plasmid pGV2850 (Zambryski, et al. (1983) EMBO J. 2:2143-2150). In the binary system, the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid. Exemplary vectors include the pBIN19 shuttle vector (Bevan (1984) Nucl. Acids Res. 12:8711-8721) and the non-oncogenic Ti plasmid pAL4404 (Hoekema, et al. (1983) Nature 303:179-180).

[0057] Promoters used in plant expression systems are typically derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV).

[0058] In mammalian cells the recombinant expression vector may be a plasmid. Alternatively, a recombinant expression vector may be a virus, or a portion thereof, which allows for expression of a nucleic acid introduced into the viral nucleic acid. For example, replication-defective retroviruses, adenoviruses and adeno-associated viruses may be used. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses may be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include, but are not limited to, pLJ, pZIP, pWE and pEM which are well-known to those skilled in the art. Examples of suitable packaging virus lines include, but are not limited to, ΨCrip, ΨCre, Ψ2 and ΨAm. The genome of adenovirus may be manipulated such that it encodes and expresses ATM kinase but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (Berkner, et al. (1988) BioTechniques 6:616; Rosenfeld, et al. (1991) Science 252:431-434; Rosenfeld, et al. (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well-known to those skilled in the art. Alternatively, an adeno-associated virus vector such as that taught by Tratschin, et al. ((1985) Mol. Cell. Biol. 5:3251-3260) may be used to express ATM kinase.

[0059] In mammalian expression systems, the regulatory sequences are often provided by the viral genome. Commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For example, the human cytomegalovirus IE promoter (Boshart, et al. (1985) Cell 41:521-530), HSV-Tk promoter (McKnight, et al. (1984) Cell 37:253-262) and β-actin promoter (Ng, et al. (1985) Mol. Cell. Biol. 5:2720-2732) may be useful in the expression of ATM kinase in mammalian cells. Alternatively, the regulatory sequences of the recombinant expression vector may direct expression of ATM kinase preferentially in a particular cell type, i.e., tissue-specific regulatory elements can be used. Examples of tissue-specific promoters which may be used include, but are not limited to, the albumin promoter (liver-specific; Pinkert, et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji, et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci USA 86:5473-5477), pancreas-specific promoters (Edlund, et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316; EP 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Camper and Tilghman (1989) Genes Dev. 3:537-546).

[0060] When the host cell is from an insect (e.g., Spodoptera frugiperda cells), expression vectors such as the baculovirus expression vector (e.g., vectors derived from Autographa californica MNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236) may be employed to express ATM kinase. In general, a baculovirus expression vector comprises a baculovirus genome containing nucleic acid sequences encoding ATM kinase inserted into the polyhedrin gene at a position ranging from the polyhedrin transcriptional start signal to the ATG start site and under the transcriptional control of a baculovirus polyhedrin promoter.

[0061]Escherichia coli is the most common prokaryotic expression system. Exemplary E. coli strains include W3110 (ATCC 27325), E. coli B, E. coli X1776 (ATCC 31537), and E. coli 294 (ATCC 31446). E. coli is typically transformed using pBR322 (Bolivar, et al. (1977) Gene 2:95) and derivatives thereof.

[0062] Promoters most commonly used in recombinant prokaryotic expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang, et al. (1978) Nature 275:615; Goeddel, et al. (1979) Nature 281:544), a tryptophan (trp) promoter system (Goeddel, et al. (1980) Nucl. Acids Res. 8:4057; EP 36,776) the tac promoter (De Boer, et al. (1983) Proc. Natl. Acad. Sci. USA 80:21) and pL of bacteriophage 1. These promoters and Shine-Dalgarno sequence may be used for efficient expression of ATM kinase in prokaryotes.

[0063] ATM kinase is expressed in a cell by introducing nucleic acid sequences encoding ATM kinase into a host cell, wherein the nucleic acids are in a form suitable for expression of ATM kinase in the host cell. Alternatively, nucleic acid sequences encoding ATM kinase which are operatively-linked to regulatory sequences (e.g., promoter sequences) but without additional vector sequences may be introduced into a host cell. As used herein, a host cell is intended to include any prokaryotic or eukaryotic cell or cell line so long as the cell or cell line is not incompatible with the protein to be expressed, the selection system chosen or the fermentation system employed. Exemplary examples of mammalian cell lines include, but are not limited to, those exemplified herein as well as CHO dhfr-cells (Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220), 293 cells (Graham, et al. (1977) J. Gen. Virol. 36:59) or myeloma cells like SP2 or NSO (Galfre and Milstein (1981) Meth. Enzymol. 73(B):3-46).

[0064] Soluble ATM kinase may be produced in by a variety of non-mammalian eukaryotic cells as well, including insect (e.g,. Spodoptera frugiperda), yeast (e.g., S. cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Kluveromyces lactis, Hansenula Polymorpha and Candida albicans, and fungal cells (Neurospora crassa, Aspergillus nidulins, Aspergillus fumigatus).

[0065] Nucleic acid sequences encoding ATM kinase may be introduced into a host cell by standard techniques for transforming cells. Transformation or transfection are intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, polyethylene glycol-mediated transformation, viral infection, Agrobacterium-mediated transformation, cell fusion, and ballistic bombardment. Suitable methods for transforming host cells may be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)) and other laboratory manuals.

[0066] The number of host cells transformed with a nucleic acid sequence encoding ATM kinase will depend, at least in part, upon the type of recombinant expression vector used and the type of transformation technique used. Nucleic acids may be introduced into a host cell transiently, or more typically, for long-term expression of ATM kinase, the nucleic acid is stably integrated into the genome of the host cell or remains as a stable episome in the host cell. Plasmid vectors introduced into mammalian cells are typically integrated into host cell DNA at only a low frequency. In order to identify these integrants, a gene that contains a selectable marker (e.g., drug resistance) is generally introduced into the host cells along with the nucleic acids of interest. Preferred selectable markers include those which confer resistance to certain drugs, such as G418 and hygromycin. Selectable markers may be introduced on a separate plasmid from the nucleic acids of interest or introduced on the same plasmid. Host cells transfected with nucleic acid sequences encoding ATM kinase (e.g., a recombinant expression vector) and a gene for a selectable marker may be identified by selecting for cells using the selectable marker. For example, if the selectable marker encodes a gene conferring neomycin resistance, host cells which have taken up nucleic acid may be selected with G418 resistance. Cells that have incorporated the selectable marker gene will survive, while the other cells die.

[0067] A host cell transformed with nucleic acid sequences encoding ATM kinase may be further transformed with one or more nucleic acids which serve as the target for ATM kinase.

[0068] Nucleic acid sequences encoding ATM kinase may be introduced into cells growing in culture in vitro by conventional transformation techniques (e.g., calcium phosphate precipitation, DEAE-dextran transfection, electroporation, etc.). Nucleic acids may also be transferred into cells in vivo, for example by application of a delivery mechanism suitable for introduction of nucleic acid into cells in vivo, such as retroviral vectors (see e.g., Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Kay, et al. (1992) Hum. Gene Ther. 3:641-647), adenoviral vectors (see e.g., Rosenfeld (1992) Cell 68:143-155; Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816), receptor-mediated DNA uptake (see e.g., Wu and Wu (1988) J. Biol. Chem. 263:14621; Wilson, et al. (1992) J. Biol. Chem. 267:963-967; U.S. Pat. No. 5,166,320), direct injection of DNA uptake (see e.g., Acsadi, et al. (1991) Nature 334:815-818; Wolff, et al. (1990) Science 247:1465-1468) or particle bombardment (see e.g., Cheng, et al. (1993) Proc. Natl. Acad. Sci. USA 90:4455-4459; Zelenin, et al. (1993) FEBS Let. 315:29-32).

[0069] Nucleic acid sequences encoding ATM kinase may be transferred into a fertilized oocyte of a non-human animal to create a transgenic animal which expresses ATM kinase in one or more cell types. A transgenic animal is an animal having cells that contain a transgene, wherein the transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic, stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. Exemplary examples of non-human animals include, but are not limited to, mice, goats, sheep, pigs, cows or other domestic farm animals. Such transgenic animals are useful, for example, for large-scale production of ATM kinase (gene pharming) or for basic research investigations.

[0070] A transgenic animal may be created, for example, by introducing a nucleic acid sequence encoding ATM kinase, typically linked to appropriate regulatory sequences, such as a constitutive or tissue-specific enhancer, into the male pronuclei of a fertilized oocyte, e.g., by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Intron sequences and polyadenylation signals may also be included in the transgene to increase the efficiency of expression of the transgene. Methods for generating transgenic animals, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. A transgenic founder animal may be used to breed additional animals carrying the transgene. Transgenic animals carrying a transgene encoding ATM kinase may further be bred to other transgenic animals carrying other transgenes, e.g., p53.

[0071] Once produced, the ATM kinase may be recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysates when directly expressed without a secretory signal. When ATM kinase is expressed in a recombinant cell other than one of human origin, the ATM kinase is completely free of proteins or polypeptides of human origin. However, it is necessary to purify ATM kinase from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to ATM kinase. As a first step, the culture medium or lysate is centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. The ATM kinase may then be purified from the soluble protein fraction. ATM kinase thereafter is purified from contaminant soluble proteins and polypeptides, as exemplified herein or with, for example, the following suitable purification procedures: by fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; ligand affinity chromatography, and protein A Sepharose columns to remove contaminants such as IgG.

[0072] A further aspect of the invention provides a cell-based method of identifying agents which modulate activation of ATM kinase. As indicated, ATM kinase in an unperturbed cell is in an inactive state. Upon DNA damage, ATM kinase is autophosphorylated at Ser¹⁹⁸¹ and converted to an active state. Therefore, an agent which modulates this autophosphorylation (i.e., activation) event may be identified in a screening assay by contacting a cell producing full-length ATM kinase or a fragment thereof retaining ATM kinase activity with an agent and determining the phosphorylation state of Ser¹⁹⁸¹. Activation of ATM kinase may be modulated by blocking, inhibiting or decreasing activation (i.e., antagonizing) as well as activating, stimulating, or increasing activation (i.e., agonizing). A typical screening assay for antagonists comprises contacting an unperturbed cell with an agent, exposing said cell to a DNA damaging agent and determining whether said agent blocks or inhibits activation of ATM kinase. Enhancers of ATM kinase activation may also be identified in this assay as agents which increase the rate or amount of ATM kinase activation following DNA damage. A typical screening assay for agonists comprises contacting an unperturbed cell with an agent and determining whether said agent stimulates activation of ATM kinase. Methods for identifying the activation state of ATM kinase in a cell are provided herein. In a preferred embodiment, activation of ATM kinase is identified by determining the phosphorylation state of Ser¹⁹⁸¹ using antibodies which specifically recognize the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase.

[0073] A further aspect of the invention provides a cell-free method of identifying agents which modulate ATM kinase activity. Soluble ATM kinase protein produced by the method disclosed herein is isolated as a monomer in an active state with an unphosphorylated Ser¹⁹⁸¹ and upon addition of a phosphate donor, autophosphorylates Ser¹⁹⁸¹ in the absence of DNA/DNA damage. Therefore, a typical screening assay using soluble ATM kinase protein comprises contacting soluble ATM kinase with an agent, exposing the soluble ATM kinase to a phosphate donor such as ATP, and detecting ATM kinase activity via autophosphorylation. The assay is carried out under suitable assay conditions for autophosphorylation, such as those exemplified herein. The phosphate donor may be added with or after the agent. It is preferred that autophosphorylation is detected using an antibody which specifically recognizes the phosphorylation state of Ser¹⁹⁸¹ of ATM kinase. Agents which antagonize ATM kinase activity are useful as radiosensitizers or chemosensitizers in the treatment of a wide variety of human tumors.

[0074] Agents which may be screened using the screening assays provided herein encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Agents may also be found among biomolecules including peptides, antibodies, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0075] Agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.

[0076] Alternatively, the antibodies of this invention may be used for the design and synthesis of either peptide or non-peptide compounds (mimetics) specific to ATM kinase (see, e.g., Saragovi, et al (1991) Science 253:792-795).

[0077] A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, and the like may be used. The mixture of components may be added in any order that provides for the requisite binding.

[0078] Alternatively, the soluble ATM kinase provided herein may be used to generate a crystal structure of ATM kinase. Once the three-dimensional structure of ATM kinase is determined, a potential agent (antagonist or agonist) can be examined through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack, et al. (1997) Folding & Design 2:27-42). This procedure can include computer fitting of potential agents to ATM kinase to ascertain how well the shape and the chemical structure of the potential ligand will complement or interfere with, the binding of ATM kinase domain with a substrate. Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of the agent. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential agent will be since these properties are consistent with a tighter binding constraint. Furthermore, the more specificity in the design of a potential agent the more likely that the agent will not interfere with related mammalian proteins. This will minimize potential side-effects due to unwanted interactions with other proteins.

[0079] The invention is described in greater detail by the following non-limiting example.

EXAMPLE 1

[0080] Cell Culture, Immunofluorescence

[0081] 293T cells, HeLa cells and 1070SK primary human foreskin fibroblasts (HFF) (>passage 20, ATCC) were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). GM00637 and GM09607 fibroblasts were grown in DMEM containing 15% FBS. GM00536 lymphoblast cells were grown in RPMI supplemented with 10% fetal calf serum. 293T cells were transfected using FUGENE™ (Roche, Indianapolis. Ind.) and HeLa cells, GM00637 and GM09607 with LIPOFECTAMINE™ (INVITROGEN™ Corp., Carlsbad, Calif.). Metabolic labeling was performed by pre-equilibrating cells in phosphate-free media for three hours prior to the addition of 0.5 mCi/ml ³²P orthophosphate (PerkinElmer Life Sciences Inc., Boston, Mass.) for 30 minutes. Inhibition of DNA synthesis and analysis of G2/M checkpoint after irradiation was assessed using well-known methods (e.g., Xu, et al. (2001) Mol. Cell. Biol. 21:3445-3450). Wortmannin was added to samples on ice 15 minutes prior to reactions and 5 μM manganese and 1 μg sonicated calf thymus DNA were present or absent. Proteins were crosslinked by incubating cells in formaldehyde/phosphate-buffered saline (PBS) for 10 minutes at room temperature. Formaldehyde was washed out using PBS containing 100 mM glycine and immunoprecipitation was then performed. Hypotonic swelling was performed in PBS containing 0.45% glucose (w/v) and 1% FBS with the NaCl concentration reduced to either 50 mM or 100 mM. For immunofluoresence experiments, HFF grown on glass slides were fixed in 50% methanol/50% acetone for two hours at −20° C. Cells were incubated with primary antibodies at 1/1000 (H2AXγ, Upstate, Charlottesville, Va.) and secondary antibodies at 1/500 (Cy3 anti-mouse and FITC anti-rabbit; Jackson ImmunoResearch Laboratories, Inc., West Grove. Pa.) in PBS, 10% FBS for one hour.

EXAMPLE 2

[0082] Plasmids and Recombinant Protein Purification

[0083] FLAG®-tagged wild-type and kinase-inactive ATM are well-known in the art (e.g., Canman, et al. (1998) Science 281:1677-1679). Wild-type, FLAG®-tagged ATM was mutated using the QUIKCHANGE® site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). The I-SceI expression plasmid is well-known in the art (Rouet, et al. (1994) Proc. Natl. Acad. Sci. USA 91:6064-6068). GST fusion proteins were expressed in BL21 from pGEX-4T-1 (Amersham Pharmacia Biotech, Uppsala, Sweden). Fusion proteins were purified and GST pull-down experiments performed by binding to glutathione-SEPHAROSE® beads (Sigma-Aldrich, St. Louis, Mo.) in PBS, 0.5% NP-40, 1 mM AEBSF and 1 mM DTT. Bound proteins were washed five times in the same buffer and eluted with 20 mM glutathione in 50 mM Tris/HCl, pH 8.0. ATM kinase domain was expressed from pET28 (NOVAGEN®, Inc., Madison, Wis.). Recombinant ATM with N-terminal 6×-His and FLAG® tags was expressed from pYES2 (INVITROGEN™, Carlsbad, Calif.) in JEL1, a protease-deficient S. cerevisiae strain that overexpresses the transcription factor GAL4 driven by the GAL1 promoter (Lindsley and Wang (1993) J. Biol. Chem. 268:8096-8104). Following induction in 2% galactose for 16 hours yeast were lysed in 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 0.4 μM aprotinin, 1 mM AEBSF, 1×soy trypsin inhibitor (Roche, Indianapolis. Ind.), 1.5 mM pepstatin and 42 μM leupeptin by three passages through a French press. Lysates were cleared by centrifugation at 35,000 rpm in a 45Ti centrifuge (Beckman COULTER®, Inc., Fullerton, Calif.). For anti-FLAG® M2 sepharose (Sigma-Aldrich, St. Louis, Mo.) affinity purification TWEEN® 20 and NP-40 were added to 1% and 0.5% respectively. FLAG® ATM was eluted in 100 μg/ml FLAG® peptide (Sigma-Aldrich, St. Louis, Mo.) in buffer A: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% TWEEN® 20, 0.5% NP-40, 50 mM NaF, 1 mM AEBSF, and 1×protease inhibitor mixture (Roche, Indianapolis. Ind.). Size-exclusion chromatography was performed using a SUPERDEX® 200HR 10/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden) in 50 mM sodium phosphate, pH 8.0 containing 150 mM NaCl. For Ni-NTA affinity purification, 20 mM imidazole was added to the lysates. 6×-His ATM was eluted from the nickel beads in 500 mM imidazole, pH 5.0, 300 mM NaCl and 2 mM EDTA.

EXAMPLE 3

[0084] Immunoprecipitation, in Vitro ATM Kinase Assays, and Peptide Mapping

[0085] Mammalian cell extracts were prepared in buffer A. Cleared supernatants were immunoprecipitated with anti-FLAG® M2 sepharose or anti-ATM D1611 (Alligood, et al. (2000) Hybridoma 19:317-321) and protein A/G agarose. Beads were washed twice with buffer A and twice with RIPA buffer. Co-immunoprecipitation was performed in buffer B: 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% TWEEN® 20, 0.2% NP-40, 50 mM NaF, 1 mM AEBSF, and 1×protease inhibitor mixture (Roche, Indianapolis. Ind.).

[0086] Ionizing irradiation induces phosphorylation of ATM at a single amino acid residue. The residue and consequence of ATM phosphorylation were examined by transiently transfecting 293T cells with either FLAG®-tagged wild-type or kinase-inactive ATM and metabolically labeling with ³²P-orthophosphate. The amount of radioactive orthophosphate incorporated into transfected wild-type ATM thirty minutes after exposure of cells to 10 Gy IR was approximately five-fold greater than that seen in unirradiated cells. In contrast, no such increase was observed following irradiation of cells that had been transfected with kinase-inactive ATM. Similarly, the amount of phosphate incorporated into endogenous ATM in immortalized lymphoblasts was markedly increased following IR. Short labeling periods were required for these experiments since exposure of cells to these amounts of radioactive orthophosphate for longer than thirty minutes damages DNA and obscures differences between irradiated and unirradiated cells (Lim, et al. (2000) Nature 404:613-617; Siliciano, et al. (1997) Genes Dev. 11:3471-3481).

[0087] Incorporation of radioactive phosphate into ATM also occurs in in vitro assays of ATM kinase activity (Canman, et al. (1998) Science 281:1677-1679; Kim, et al. (1999) J. Biol. Chem. 274:37538-37543). For in vitro kinase assays, beads were washed twice with buffer A, twice with buffer A containing 0.5 M LiCl, and twice with kinase buffer: 20 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl₂ and 10 mM MnCl₂. ATM kinase reactions were performed at 30° C. for five minutes in 50 μl of kinase buffer containing 10 μCi of [γ-³²P] ATP and 1 μg of GST-fusion substrate. In vitro phosphorylation of ATM was not seen with the kinase-inactive ATM protein; was inhibited by exposure to 30 nM of the PI-3K inhibitor wortmannin; was dependent on the addition of the divalent cation manganese; and was not dependent on addition of exogenous DNA. Identical properties are characteristic of ATM in its phosphorylation of target substrates: a concentration of 30 nM wortmannin effectively inhibits ATM kinase activity (Sarkaria, et al. (1998) Cancer Res. 58:4375-4382); its activity requires the presence of manganese (Canman, et al. (1998) Science 281:1677-1679; Kim, et al. (1999) J. Biol. Chem. 274:37538-37543); and exogenous DNA does not enhance its activity after immunoprecipitation from cells (Kim, et al. (1999) J. Biol. Chem. 274:37538-37543). Together with the observation that ATM kinase activity was required for the increase in ATM phosphorylation seen during metabolic labeling of cells, these findings indicate that ATM phosphorylates itself.

[0088] The site of phosphorylation in ATM was identified by radioactively labeling ATM protein, digesting with trypsin, and analyzing tryptic phosphopeptides by sequential two-dimensional (2-D) electrophoresis and chromatography. Tryptic digestion of FLAG® ATM immobilized on nitrocellulose, 2-D resolution (electrophoresis and chromatography), manual Edman degradation and V8 digestion of peptides isolated from thin-layer chromatography plates were performed using well-known methods (Meisenhelder et al. Current Protocols in Molecular Biology. Ausubel, F. M. et al. (eds.) (1999)). A single de novo phosphorylated peptide was identified in both transfected FLAG®-tagged ATM and in endogenous ATM isolated from irradiated cells. Since the increase in phosphorylation of this peptide was seen in vivo with wild-type, but not kinase-inactive ATM, this event is consistent with IR-inducible ATM autophosphorylation.

[0089] The ATM amino acid residue phosphorylated following IR was determined. The phosphopeptide identified by thin layer chromatography was isolated and subjected to acid hydrolysis. Resolution of the labeled phosphoamino acid by 2-D electrophoresis with unlabeled phosphoamino acid markers revealed phosphoserine. There are 121 predicted tryptic peptides in ATM containing a single phosphoserine residue and many additional peptides containing two or more. Direct identification of the phosphopeptide by automated sequencing and mass spectrometry failed, at least in part due to difficulties in obtaining sufficient quantities of material.

[0090] The predicted migrations of tryptic serine-containing phosphopeptides in ATM eliminated many candidates. Forty-one tryptic serine phosphopeptides, whose theoretical migration satisfied criteria for that of the principal peptide at pH 1.9 and pH 4.72, were synthesized and subjected to 2-D electrophoresis and chromatography along with tryptic digests of radioactively labeled ATM. The theoretical and observed migrations of the 41 peptides were coincident at both pH's but this nonetheless failed to identify the radiolabeled phosphopeptide.

[0091] Many kinases are activated by phosphorylation within their kinase domains (Johnson and Noble (1996) Cell 85:149-158), therefore, each serine in the kinase domain, as well as several other Ser-Gln sequences in ATM, were subjected to site-directed mutagenesis. Individual serines in wild-type, FLAG®-tagged ATM were mutated to glycine or alanine and the mutants were expressed, immunoprecipitated and allowed to autophosphorylate in vitro. The labeled, mutant ATM proteins were then digested with trypsin and the resulting tryptic phosphopeptides were resolved by 2-D mapping. Surprisingly, the major phosphopeptide was still detected on 2-D maps of each ATM mutant, all of which retained kinase activity. Therefore, none of the serines in the kinase domain of ATM are required for its kinase activity.

[0092] The major ATM phosphopeptide was identified by use of secondary proteolytic and chemical cleavage of the primary phosphopeptide. After in vivo radioactive labeling of ATM protein and purification of the phosphopeptide from the thin layer chromatography plate, the purified tryptic phosphopeptide was digested with V8 protease. The appearance of a peptide with a new mobility on 2-D maps dictated the presence of a glutamic acid in the peptide. Furthermore, an unchanged chromatographic mobility after cyanogen bromide treatment of the purified tryptic phosphopeptide in formic acid indicated that it did not contain methionine.

[0093] Manual Edman degradation was then performed on both the tryptic serine phosphopeptide and the derived V8/tryptic phosphopeptide. Resolution of the Edman degradation products revealed a secondary spot in each cycle suggesting a derivatized peptide. If the peptide being treated in the Edman reaction contains a C-terminal lysine, the phenylisothiocyanate treatment followed by acid treatment causes a derivatization of the lysine E-amino group (Meisenhelder et al. Current Protocols in Molecular Biology. Ausubel, F. M. et al. (eds.) (1999)). Thus, the manual Edman reactions indicated that the phosphopeptide of interest had a C-terminal lysine. The second cycle of Edman degradation resulted in the generation of free phosphate from the V8/tryptic phosphopeptide. Thus, the phosphorylated serine is two amino acid residues to the C-terminal side of a glutamic acid residue. Further cycles of Edman degradation were required to release the phosphate from the original tryptic phosphopeptide, with generation of some free phosphate at cycle eight and substantial release at cycle nine. Thus, the phosphorylated serine in the larger peptide was eight or nine amino acid residues to the C-terminal side of either a lysine or arginine residue.

[0094] Although inefficiencies in the manual Edman degradation process cause difficulties when more than five or six cycles are required for amino acid release (Meisenhelder et al. Current Protocols in Molecular Biology. Ausubel, F. M. et al. (eds.) (1999)), a partial sequence was obtained for the major serine phosphopeptide of Xaa₅₋₆-Glu-Xaa-Ser-Xaa_(n)-Lys (SEQ ID NO: 3) (where Xaa is any amino acid other than Met, the N-terminus contains 5-6 residues, and the sequence preceding the C-terminal Lys is of unknown length, n). The only tryptic peptide in ATM which meets these sequence requirements is the 19 residue peptide 1974-Ser-Leu-Ala-Phe-Glu-Glu-Gly-Ser-Gln-Ser-Thr-Thr-Ile-Ser-Ser-Leu-Ser-Glu-Lys-1992 (SEQ ID NO: 4). In addition, since V8 protease does not cleave at every glutamic acid in a peptide sequence, the adjacent residues Glu¹⁹⁷⁸ and Glu¹⁹⁷⁹ explain the release of free phosphate at both cycles two and three in the manual Edman degradation of the V8/tryptic phosphopeptide. The V8 phosphopeptide was likely a mixture of two peptides that were unresolved on the 2-D map at pH 1.9, one of which had been cleaved at Glu¹⁹⁷⁸ and one at Glu¹⁹⁷⁹. It is noted that V8 did not digest the tryptic phosphopeptide at Glu¹⁹⁹¹ since the derivatization of Lys¹⁹⁹² in the manual Edman degradation of the V8/tryptic phosphopeptide was seen. Ser¹⁹⁸¹ in ATM is conserved in mouse and Xenopus ATM, but is not found in suspected homologues of less complex metazoans and is located in the N-terminus of the FAT domain, a region of approximately 500 amino acid residues with some conservation across the PI-3K family of kinases including Frap, ATM and Trapp (Bosotti, el al. (2000) Trends Biochem. Sci. 25:225-227). It is also noted that this is an ‘Ser-Gln’ site, thus indicating either an autophosphorylation event or phosphorylation by an ATM family member.

EXAMPLE 4

[0095] Western Blotting and Antisera Production

[0096] Western blotting was performed with anti-FLAG® M5 (Sigma-Aldrich, St. Louis, Mo.), anti-ATM MAT3, anti-GST (Amersham Pharmacia Biotech, Uppsala, Sweden) or anti-6×-His (Sigma-Aldrich, St. Louis, Mo.). Anti-Ser¹⁹⁸¹ and anti-Ser¹⁹⁸¹-P specific antibodies were generated by immunizing rabbits with KLH-conjugated synthetic peptides Ser-Leu-Ala-Phe-Glu-Glu-Gly-Ser-Gln-Ser-Thr-Thr-Ile-Ser-Ser (SEQ ID NO: 5) (three animals) and Ser-Leu-Ala-Phe-Glu-Glu-Gly-Ser-Pro-Gln-Ser-Thr-Thr-Ile-Ser-Ser (SEQ ID NO: 6) (6 animals), respectively (Rockland Immunochemicals, Gilbertsville, Pa.).

EXAMPLE 5

[0097] Autophosphorylation

[0098] Autophosphorylation of Ser¹⁹⁸¹ was determined using glutathione-S-transferase (GST) fusion proteins containing various lengths of ATM as substrates of the ATM kinase in in vitro kinase reactions. A polypeptide containing the appropriate serine residue at 1981 was an excellent in vitro substrate, whereas a Ser¹⁹⁸¹→Ala mutant was not phosphorylated by ATM whether the read-out was incorporation of radioactive phosphate or binding by the α-Ser¹⁹⁸¹-P antibody. In addition, wortmannin concentrations of 20 μM or more effectively inhibited phosphorylation of Ser¹⁹⁸¹ following irradiation of human diploid fibroblasts. This concentration of wortmannin inhibits both ATM and DNA-dependent protein kinase (DNA-PK), but not ataxia- and Rad3-related protein (ATR), in vivo (Sarkaria, et al. (1998) Cancer Res. 58:4375-4382). To exclude DNA-PK as a responsible kinase, phosphorylation of Ser¹⁹⁸¹ was determined in a cell line lacking DNA-PK activity. The kinetics and levels of IR-induced phosphorylation of Ser¹⁹⁸¹ were identical to those in a similar cell line containing DNA-PK activity.

[0099] In vivo experiments demonstrated that optimal phosphorylation of ATM was dependent on the presence of active ATM kinase, however, the cells used contained endogenous wild-type ATM. Thus, even the kinase-inactive mutant of ATM was phosphorylated to some extent in these cells. The importance of ATM kinase activity in the phosphorylation of Ser¹⁹⁸¹ was further analyzed using constructs encoding wild-type, kinase-inactive, and Ser¹⁹⁸¹→Ala ATM transfected into A-T cells such that the only potential source of ATM kinase activity was the transgene being utilized. All of these constructs were expressed at similar levels in A-T cells, but only wild-type ATM was recognized by α-Ser¹⁹⁸¹-P, and its binding was increased several-fold within 30 minutes after exposure to 10 Gy IR. Although some phosphorylation of kinase-inactive ATM was observed following transfection into 293T cells, which contain endogenous ATM activity, transfection into a cell line that lacked ATM kinase activity showed no detectable phosphorylation on Ser¹⁹⁸¹. Therefore, phosphorylation of Ser¹⁹⁸¹ depends on the activity of the ATM kinase itself and the phosphorylation of transfected kinase-inactive ATM must occur in trans.

[0100] Mutating Ser¹⁹⁸¹ did not abrogate ATM kinase activity in vitro, but conferred dominant inhibitory activity in cells. Expression vectors encoding wild-type and Ser¹⁹⁸¹→Ala ATM proteins were transfected into A-T fibroblasts and kinase activity was assessed by in vitro kinase assays performed with the immunoprecipitated ATM proteins. Both wild-type and Ser¹⁹⁸¹→Ala mutant ATM exhibited in vitro kinase activity directed against a peptide containing the Ser¹⁵ target sequence in p53. To explore the in vivo activity of the Ser 1981→Ala mutant, nucleic acid sequences encoding ATM kinase were introduced into HeLa cells and the integrity of their ATM-dependent, IR-induced G2 and S-phase checkpoints was examined. Despite its kinase activity in the in vitro assay, Ser¹⁹⁸¹→Ala ATM mimicked kinase-inactive ATM in inhibiting both the IR-induced G2 checkpoint and S-phase replication arrest. Consistent with its abrogation of the IR-induced cell cycle checkpoints, the transfected Ser¹⁹⁸¹→Ala mutant also blocked the IR-induced phosphorylation of endogenous ATM protein on Ser¹⁹⁸¹. Thus, whereas phosphorylation of ATM on Ser¹⁹⁸¹ does not appear to be required for kinase activity in vitro, expression of Ser¹⁹⁸¹→Ala ATM effectively inhibits the cellular activities of endogenous ATM in a dominant-inhibitory manner.

1 6 1 3056 PRT Homo sapiens 1 Met Ser Leu Val Leu Asn Asp Leu Leu Ile Cys Cys Arg Gln Leu Glu 1 5 10 15 His Asp Arg Ala Thr Glu Arg Lys Lys Glu Val Glu Lys Phe Lys Arg 20 25 30 Leu Ile Arg Asp Pro Glu Thr Ile Lys His Leu Asp Arg His Ser Asp 35 40 45 Ser Lys Gln Gly Lys Tyr Leu Asn Trp Asp Ala Val Phe Arg Phe Leu 50 55 60 Gln Lys Tyr Ile Gln Lys Glu Thr Glu Cys Leu Arg Ile Ala Lys Pro 65 70 75 80 Asn Val Ser Ala Ser Thr Gln Ala Ser Arg Gln Lys Lys Met Gln Glu 85 90 95 Ile Ser Ser Leu Val Lys Tyr Phe Ile Lys Cys Ala Asn Arg Arg Ala 100 105 110 Pro Arg Leu Lys Cys Gln Glu Leu Leu Asn Tyr Ile Met Asp Thr Val 115 120 125 Lys Asp Ser Ser Asn Gly Ala Ile Tyr Gly Ala Asp Cys Ser Asn Ile 130 135 140 Leu Leu Lys Asp Ile Leu Ser Val Arg Lys Tyr Trp Cys Glu Ile Ser 145 150 155 160 Gln Gln Gln Trp Leu Glu Leu Phe Ser Val Tyr Phe Arg Leu Tyr Leu 165 170 175 Lys Pro Ser Gln Asp Val His Arg Val Leu Val Ala Arg Ile Ile His 180 185 190 Ala Val Thr Lys Gly Cys Cys Ser Gln Thr Asp Gly Leu Asn Ser Lys 195 200 205 Phe Leu Asp Phe Phe Ser Lys Ala Ile Gln Cys Ala Arg Gln Glu Lys 210 215 220 Ser Ser Ser Gly Leu Asn His Ile Leu Ala Ala Leu Thr Ile Phe Leu 225 230 235 240 Lys Thr Leu Ala Val Asn Phe Arg Ile Arg Val Cys Glu Leu Gly Asp 245 250 255 Glu Ile Leu Pro Thr Leu Leu Tyr Ile Trp Thr Gln His Arg Leu Asn 260 265 270 Asp Ser Leu Lys Glu Val Ile Ile Glu Leu Phe Gln Leu Gln Ile Tyr 275 280 285 Ile His His Pro Lys Gly Ala Lys Thr Gln Glu Lys Gly Ala Tyr Glu 290 295 300 Ser Thr Lys Trp Arg Ser Ile Leu Tyr Asn Leu Tyr Asp Leu Leu Val 305 310 315 320 Asn Glu Ile Ser His Ile Gly Ser Arg Gly Lys Tyr Ser Ser Gly Phe 325 330 335 Arg Asn Ile Ala Val Lys Glu Asn Leu Ile Glu Leu Met Ala Asp Ile 340 345 350 Cys His Gln Val Phe Asn Glu Asp Thr Arg Ser Leu Glu Ile Ser Gln 355 360 365 Ser Tyr Thr Thr Thr Gln Arg Glu Ser Ser Asp Tyr Ser Val Pro Cys 370 375 380 Lys Arg Lys Lys Ile Glu Leu Gly Trp Glu Val Ile Lys Asp His Leu 385 390 395 400 Gln Lys Ser Gln Asn Asp Phe Asp Leu Val Pro Trp Leu Gln Ile Ala 405 410 415 Thr Gln Leu Ile Ser Lys Tyr Pro Ala Ser Leu Pro Asn Cys Glu Leu 420 425 430 Ser Pro Leu Leu Met Ile Leu Ser Gln Leu Leu Pro Gln Gln Arg His 435 440 445 Gly Glu Arg Thr Pro Tyr Val Leu Arg Cys Leu Thr Glu Val Ala Leu 450 455 460 Cys Gln Asp Lys Arg Ser Asn Leu Glu Ser Ser Gln Lys Ser Asp Leu 465 470 475 480 Leu Lys Leu Trp Asn Lys Ile Trp Cys Ile Thr Phe Arg Gly Ile Ser 485 490 495 Ser Glu Gln Ile Gln Ala Glu Asn Phe Gly Leu Leu Gly Ala Ile Ile 500 505 510 Gln Gly Ser Leu Val Glu Val Asp Arg Glu Phe Trp Lys Leu Phe Thr 515 520 525 Gly Ser Ala Cys Arg Pro Ser Cys Pro Ala Val Cys Cys Leu Thr Leu 530 535 540 Ala Leu Thr Thr Ser Ile Val Pro Gly Ala Val Lys Met Gly Ile Glu 545 550 555 560 Gln Asn Met Cys Glu Val Asn Arg Ser Phe Ser Leu Lys Glu Ser Ile 565 570 575 Met Lys Trp Leu Leu Phe Tyr Gln Leu Glu Gly Asp Leu Glu Asn Ser 580 585 590 Thr Glu Val Pro Pro Ile Leu His Ser Asn Phe Pro His Leu Val Leu 595 600 605 Glu Lys Ile Leu Val Ser Leu Thr Met Lys Asn Cys Lys Ala Ala Met 610 615 620 Asn Phe Phe Gln Ser Val Pro Glu Cys Glu His His Gln Lys Asp Lys 625 630 635 640 Glu Glu Leu Ser Phe Ser Glu Val Glu Glu Leu Phe Leu Gln Thr Thr 645 650 655 Phe Asp Lys Met Asp Phe Leu Thr Ile Val Arg Glu Cys Gly Ile Glu 660 665 670 Lys His Gln Ser Ser Ile Gly Phe Ser Val His Gln Asn Leu Lys Glu 675 680 685 Ser Leu Asp Arg Cys Leu Leu Gly Leu Ser Glu Gln Leu Leu Asn Asn 690 695 700 Tyr Ser Ser Glu Ile Thr Asn Ser Glu Thr Leu Val Arg Cys Ser Arg 705 710 715 720 Leu Leu Val Gly Val Leu Gly Cys Tyr Cys Tyr Met Gly Val Ile Ala 725 730 735 Glu Glu Glu Ala Tyr Lys Ser Glu Leu Phe Gln Lys Ala Asn Ser Leu 740 745 750 Met Gln Cys Ala Gly Glu Ser Ile Thr Leu Phe Lys Asn Lys Thr Asn 755 760 765 Glu Glu Phe Arg Ile Gly Ser Leu Arg Asn Met Met Gln Leu Cys Thr 770 775 780 Arg Cys Leu Ser Asn Cys Thr Lys Lys Ser Pro Asn Lys Ile Ala Ser 785 790 795 800 Gly Phe Phe Leu Arg Leu Leu Thr Ser Lys Leu Met Asn Asp Ile Ala 805 810 815 Asp Ile Cys Lys Ser Leu Ala Ser Phe Ile Lys Lys Pro Phe Asp Arg 820 825 830 Gly Glu Val Glu Ser Met Glu Asp Asp Thr Asn Gly Asn Leu Met Glu 835 840 845 Val Glu Asp Gln Ser Ser Met Asn Leu Phe Asn Asp Tyr Pro Asp Ser 850 855 860 Ser Val Ser Asp Ala Asn Glu Pro Gly Glu Ser Gln Ser Thr Ile Gly 865 870 875 880 Ala Ile Asn Pro Leu Ala Glu Glu Tyr Leu Ser Lys Gln Asp Leu Leu 885 890 895 Phe Leu Asp Met Leu Lys Phe Leu Cys Leu Cys Val Thr Thr Ala Gln 900 905 910 Thr Asn Thr Val Ser Phe Arg Ala Ala Asp Ile Arg Arg Lys Leu Leu 915 920 925 Met Leu Ile Asp Ser Ser Thr Leu Glu Pro Thr Lys Ser Leu His Leu 930 935 940 His Met Tyr Leu Met Leu Leu Lys Glu Leu Pro Gly Glu Glu Tyr Pro 945 950 955 960 Leu Pro Met Glu Asp Val Leu Glu Leu Leu Lys Pro Leu Ser Asn Val 965 970 975 Cys Ser Leu Tyr Arg Arg Asp Gln Asp Val Cys Lys Thr Ile Leu Asn 980 985 990 His Val Leu His Val Val Lys Asn Leu Gly Gln Ser Asn Met Asp Ser 995 1000 1005 Glu Asn Thr Arg Asp Ala Gln Gly Gln Phe Leu Thr Val Ile Gly 1010 1015 1020 Ala Phe Trp His Leu Thr Lys Glu Arg Lys Tyr Ile Phe Ser Val 1025 1030 1035 Arg Met Ala Leu Val Asn Cys Leu Lys Thr Leu Leu Glu Ala Asp 1040 1045 1050 Pro Tyr Ser Lys Trp Ala Ile Leu Asn Val Met Gly Lys Asp Phe 1055 1060 1065 Pro Val Asn Glu Val Phe Thr Gln Phe Leu Ala Asp Asn His His 1070 1075 1080 Gln Val Arg Met Leu Ala Ala Glu Ser Ile Asn Arg Leu Phe Gln 1085 1090 1095 Asp Thr Lys Gly Asp Ser Ser Arg Leu Leu Lys Ala Leu Pro Leu 1100 1105 1110 Lys Leu Gln Gln Thr Ala Phe Glu Asn Ala Tyr Leu Lys Ala Gln 1115 1120 1125 Glu Gly Met Arg Glu Met Ser His Ser Ala Glu Asn Pro Glu Thr 1130 1135 1140 Leu Asp Glu Ile Tyr Asn Arg Lys Ser Val Leu Leu Thr Leu Ile 1145 1150 1155 Ala Val Val Leu Ser Cys Ser Pro Ile Cys Glu Lys Gln Ala Leu 1160 1165 1170 Phe Ala Leu Cys Lys Ser Val Lys Glu Asn Gly Leu Glu Pro His 1175 1180 1185 Leu Val Lys Lys Val Leu Glu Lys Val Ser Glu Thr Phe Gly Tyr 1190 1195 1200 Arg Arg Leu Glu Asp Phe Met Ala Ser His Leu Asp Tyr Leu Val 1205 1210 1215 Leu Glu Trp Leu Asn Leu Gln Asp Thr Glu Tyr Asn Leu Ser Ser 1220 1225 1230 Phe Pro Phe Ile Leu Leu Asn Tyr Thr Asn Ile Glu Asp Phe Tyr 1235 1240 1245 Arg Ser Cys Tyr Lys Val Leu Ile Pro His Leu Val Ile Arg Ser 1250 1255 1260 His Phe Asp Glu Val Lys Ser Ile Ala Asn Gln Ile Gln Glu Asp 1265 1270 1275 Trp Lys Ser Leu Leu Thr Asp Cys Phe Pro Lys Ile Leu Val Asn 1280 1285 1290 Ile Leu Pro Tyr Phe Ala Tyr Glu Gly Thr Arg Asp Ser Gly Met 1295 1300 1305 Ala Gln Gln Arg Glu Thr Ala Thr Lys Val Tyr Asp Met Leu Lys 1310 1315 1320 Ser Glu Asn Leu Leu Gly Lys Gln Ile Asp His Leu Phe Ile Ser 1325 1330 1335 Asn Leu Pro Glu Ile Val Val Glu Leu Leu Met Thr Leu His Glu 1340 1345 1350 Pro Ala Asn Ser Ser Ala Ser Gln Ser Thr Asp Leu Cys Asp Phe 1355 1360 1365 Ser Gly Asp Leu Asp Pro Ala Pro Asn Pro Pro His Phe Pro Ser 1370 1375 1380 His Val Ile Lys Ala Thr Phe Ala Tyr Ile Ser Asn Cys His Lys 1385 1390 1395 Thr Lys Leu Lys Ser Ile Leu Glu Ile Leu Ser Lys Ser Pro Asp 1400 1405 1410 Ser Tyr Gln Lys Ile Leu Leu Ala Ile Cys Glu Gln Ala Ala Glu 1415 1420 1425 Thr Asn Asn Val Tyr Lys Lys His Arg Ile Leu Lys Ile Tyr His 1430 1435 1440 Leu Phe Val Ser Leu Leu Leu Lys Asp Ile Lys Ser Gly Leu Gly 1445 1450 1455 Gly Ala Trp Ala Phe Val Leu Arg Asp Val Ile Tyr Thr Leu Ile 1460 1465 1470 His Tyr Ile Asn Gln Arg Pro Ser Cys Ile Met Asp Val Ser Leu 1475 1480 1485 Arg Ser Phe Ser Leu Cys Cys Asp Leu Leu Ser Gln Val Cys Gln 1490 1495 1500 Thr Ala Val Thr Tyr Cys Lys Asp Ala Leu Glu Asn His Leu His 1505 1510 1515 Val Ile Val Gly Thr Leu Ile Pro Leu Val Tyr Glu Gln Val Glu 1520 1525 1530 Val Gln Lys Gln Val Leu Asp Leu Leu Lys Tyr Leu Val Ile Asp 1535 1540 1545 Asn Lys Asp Asn Glu Asn Leu Tyr Ile Thr Ile Lys Leu Leu Asp 1550 1555 1560 Pro Phe Pro Asp His Val Val Phe Lys Asp Leu Arg Ile Thr Gln 1565 1570 1575 Gln Lys Ile Lys Tyr Ser Arg Gly Pro Phe Ser Leu Leu Glu Glu 1580 1585 1590 Ile Asn His Phe Leu Ser Val Ser Val Tyr Asp Ala Leu Pro Leu 1595 1600 1605 Thr Arg Leu Glu Gly Leu Lys Asp Leu Arg Arg Gln Leu Glu Leu 1610 1615 1620 His Lys Asp Gln Met Val Asp Ile Met Arg Ala Ser Gln Asp Asn 1625 1630 1635 Pro Gln Asp Gly Ile Met Val Lys Leu Val Val Asn Leu Leu Gln 1640 1645 1650 Leu Ser Lys Met Ala Ile Asn His Thr Gly Glu Lys Glu Val Leu 1655 1660 1665 Glu Ala Val Gly Ser Cys Leu Gly Glu Val Gly Pro Ile Asp Phe 1670 1675 1680 Ser Thr Ile Ala Ile Gln His Ser Lys Asp Ala Ser Tyr Thr Lys 1685 1690 1695 Ala Leu Lys Leu Phe Glu Asp Lys Glu Leu Gln Trp Thr Phe Ile 1700 1705 1710 Met Leu Thr Tyr Leu Asn Asn Thr Leu Val Glu Asp Cys Val Lys 1715 1720 1725 Val Arg Ser Ala Ala Val Thr Cys Leu Lys Asn Ile Leu Ala Thr 1730 1735 1740 Lys Thr Gly His Ser Phe Trp Glu Ile Tyr Lys Met Thr Thr Asp 1745 1750 1755 Pro Met Leu Ala Tyr Leu Gln Pro Phe Arg Thr Ser Arg Lys Lys 1760 1765 1770 Phe Leu Glu Val Pro Arg Phe Asp Lys Glu Asn Pro Phe Glu Gly 1775 1780 1785 Leu Asp Asp Ile Asn Leu Trp Ile Pro Leu Ser Glu Asn His Asp 1790 1795 1800 Ile Trp Ile Lys Thr Leu Thr Cys Ala Phe Leu Asp Ser Gly Gly 1805 1810 1815 Thr Lys Cys Glu Ile Leu Gln Leu Leu Lys Pro Met Cys Glu Val 1820 1825 1830 Lys Thr Asp Phe Cys Gln Thr Val Leu Pro Tyr Leu Ile His Asp 1835 1840 1845 Ile Leu Leu Gln Asp Thr Asn Glu Ser Trp Arg Asn Leu Leu Ser 1850 1855 1860 Thr His Val Gln Gly Phe Phe Thr Ser Cys Leu Arg His Phe Ser 1865 1870 1875 Gln Thr Ser Arg Ser Thr Thr Pro Ala Asn Leu Asp Ser Glu Ser 1880 1885 1890 Glu His Phe Phe Arg Cys Cys Leu Asp Lys Lys Ser Gln Arg Thr 1895 1900 1905 Met Leu Ala Val Val Asp Tyr Met Arg Arg Gln Lys Arg Pro Ser 1910 1915 1920 Ser Gly Thr Ile Phe Asn Asp Ala Phe Trp Leu Asp Leu Asn Tyr 1925 1930 1935 Leu Glu Val Ala Lys Val Ala Gln Ser Cys Ala Ala His Phe Thr 1940 1945 1950 Ala Leu Leu Tyr Ala Glu Ile Tyr Ala Asp Lys Lys Ser Met Asp 1955 1960 1965 Asp Gln Glu Lys Arg Ser Leu Ala Phe Glu Glu Gly Ser Gln Ser 1970 1975 1980 Thr Thr Ile Ser Ser Leu Ser Glu Lys Ser Lys Glu Glu Thr Gly 1985 1990 1995 Ile Ser Leu Gln Asp Leu Leu Leu Glu Ile Tyr Arg Ser Ile Gly 2000 2005 2010 Glu Pro Asp Ser Leu Tyr Gly Cys Gly Gly Gly Lys Met Leu Gln 2015 2020 2025 Pro Ile Thr Arg Leu Arg Thr Tyr Glu His Glu Ala Met Trp Gly 2030 2035 2040 Lys Ala Leu Val Thr Tyr Asp Leu Glu Thr Ala Ile Pro Ser Ser 2045 2050 2055 Thr Arg Gln Ala Gly Ile Ile Gln Ala Leu Gln Asn Leu Gly Leu 2060 2065 2070 Cys His Ile Leu Ser Val Tyr Leu Lys Gly Leu Asp Tyr Glu Asn 2075 2080 2085 Lys Asp Trp Cys Pro Glu Leu Glu Glu Leu His Tyr Gln Ala Ala 2090 2095 2100 Trp Arg Asn Met Gln Trp Asp His Cys Thr Ser Val Ser Lys Glu 2105 2110 2115 Val Glu Gly Thr Ser Tyr His Glu Ser Leu Tyr Asn Ala Leu Gln 2120 2125 2130 Ser Leu Arg Asp Arg Glu Phe Ser Thr Phe Tyr Glu Ser Leu Lys 2135 2140 2145 Tyr Ala Arg Val Lys Glu Val Glu Glu Met Cys Lys Arg Ser Leu 2150 2155 2160 Glu Ser Val Tyr Ser Leu Tyr Pro Thr Leu Ser Arg Leu Gln Ala 2165 2170 2175 Ile Gly Glu Leu Glu Ser Ile Gly Glu Leu Phe Ser Arg Ser Val 2180 2185 2190 Thr His Arg Gln Leu Ser Glu Val Tyr Ile Lys Trp Gln Lys His 2195 2200 2205 Ser Gln Leu Leu Lys Asp Ser Asp Phe Ser Phe Gln Glu Pro Ile 2210 2215 2220 Met Ala Leu Arg Thr Val Ile Leu Glu Ile Leu Met Glu Lys Glu 2225 2230 2235 Met Asp Asn Ser Gln Arg Glu Cys Ile Lys Asp Ile Leu Thr Lys 2240 2245 2250 His Leu Val Glu Leu Ser Ile Leu Ala Arg Thr Phe Lys Asn Thr 2255 2260 2265 Gln Leu Pro Glu Arg Ala Ile Phe Gln Ile Lys Gln Tyr Asn Ser 2270 2275 2280 Val Ser Cys Gly Val Ser Glu Trp Gln Leu Glu Glu Ala Gln Val 2285 2290 2295 Phe Trp Ala Lys Lys Glu Gln Ser Leu Ala Leu Ser Ile Leu Lys 2300 2305 2310 Gln Met Ile Lys Lys Leu Asp Ala Ser Cys Ala Ala Asn Asn Pro 2315 2320 2325 Ser Leu Lys Leu Thr Tyr Thr Glu Cys Leu Arg Val Cys Gly Asn 2330 2335 2340 Trp Leu Ala Glu Thr Cys Leu Glu Asn Pro Ala Val Ile Met Gln 2345 2350 2355 Thr Tyr Leu Glu Lys Ala Val Glu Val Ala Gly Asn Tyr Asp Gly 2360 2365 2370 Glu Ser Ser Asp Glu Leu Arg Asn Gly Lys Met Lys Ala Phe Leu 2375 2380 2385 Ser Leu Ala Arg Phe Ser Asp Thr Gln Tyr Gln Arg Ile Glu Asn 2390 2395 2400 Tyr Met Lys Ser Ser Glu Phe Glu Asn Lys Gln Ala Leu Leu Lys 2405 2410 2415 Arg Ala Lys Glu Glu Val Gly Leu Leu Arg Glu His Lys Ile Gln 2420 2425 2430 Thr Asn Arg Tyr Thr Val Lys Val Gln Arg Glu Leu Glu Leu Asp 2435 2440 2445 Glu Leu Ala Leu Arg Ala Leu Lys Glu Asp Arg Lys Arg Phe Leu 2450 2455 2460 Cys Lys Ala Val Glu Asn Tyr Ile Asn Cys Leu Leu Ser Gly Glu 2465 2470 2475 Glu His Asp Met Trp Val Phe Arg Leu Cys Ser Leu Trp Leu Glu 2480 2485 2490 Asn Ser Gly Val Ser Glu Val Asn Gly Met Met Lys Arg Asp Gly 2495 2500 2505 Met Lys Ile Pro Thr Tyr Lys Phe Leu Pro Leu Met Tyr Gln Leu 2510 2515 2520 Ala Ala Arg Met Gly Thr Lys Met Met Gly Gly Leu Gly Phe His 2525 2530 2535 Glu Val Leu Asn Asn Leu Ile Ser Arg Ile Ser Met Asp His Pro 2540 2545 2550 His His Thr Leu Phe Ile Ile Leu Ala Leu Ala Asn Ala Asn Arg 2555 2560 2565 Asp Glu Phe Leu Thr Lys Pro Glu Val Ala Arg Arg Ser Arg Ile 2570 2575 2580 Thr Lys Asn Val Pro Lys Gln Ser Ser Gln Leu Asp Glu Asp Arg 2585 2590 2595 Thr Glu Ala Ala Asn Arg Ile Ile Cys Thr Ile Arg Ser Arg Arg 2600 2605 2610 Pro Gln Met Val Arg Ser Val Glu Ala Leu Cys Asp Ala Tyr Ile 2615 2620 2625 Ile Leu Ala Asn Leu Asp Ala Thr Gln Trp Lys Thr Gln Arg Lys 2630 2635 2640 Gly Ile Asn Ile Pro Ala Asp Gln Pro Ile Thr Lys Leu Lys Asn 2645 2650 2655 Leu Glu Asp Val Val Val Pro Thr Met Glu Ile Lys Val Asp His 2660 2665 2670 Thr Gly Glu Tyr Gly Asn Leu Val Thr Ile Gln Ser Phe Lys Ala 2675 2680 2685 Glu Phe Arg Leu Ala Gly Gly Val Asn Leu Pro Lys Ile Ile Asp 2690 2695 2700 Cys Val Gly Ser Asp Gly Lys Glu Arg Arg Gln Leu Val Lys Gly 2705 2710 2715 Arg Asp Asp Leu Arg Gln Asp Ala Val Met Gln Gln Val Phe Gln 2720 2725 2730 Met Cys Asn Thr Leu Leu Gln Arg Asn Thr Glu Thr Arg Lys Arg 2735 2740 2745 Lys Leu Thr Ile Cys Thr Tyr Lys Val Val Pro Leu Ser Gln Arg 2750 2755 2760 Ser Gly Val Leu Glu Trp Cys Thr Gly Thr Val Pro Ile Gly Glu 2765 2770 2775 Phe Leu Val Asn Asn Glu Asp Gly Ala His Lys Arg Tyr Arg Pro 2780 2785 2790 Asn Asp Phe Ser Ala Phe Gln Cys Gln Lys Lys Met Met Glu Val 2795 2800 2805 Gln Lys Lys Ser Phe Glu Glu Lys Tyr Glu Val Phe Met Asp Val 2810 2815 2820 Cys Gln Asn Phe Gln Pro Val Phe Arg Tyr Phe Cys Met Glu Lys 2825 2830 2835 Phe Leu Asp Pro Ala Ile Trp Phe Glu Lys Arg Leu Ala Tyr Thr 2840 2845 2850 Arg Ser Val Ala Thr Ser Ser Ile Val Gly Tyr Ile Leu Gly Leu 2855 2860 2865 Gly Asp Arg His Val Gln Asn Ile Leu Ile Asn Glu Gln Ser Ala 2870 2875 2880 Glu Leu Val His Ile Asp Leu Gly Val Ala Phe Glu Gln Gly Lys 2885 2890 2895 Ile Leu Pro Thr Pro Glu Thr Val Pro Phe Arg Leu Thr Arg Asp 2900 2905 2910 Ile Val Asp Gly Met Gly Ile Thr Gly Val Glu Gly Val Phe Arg 2915 2920 2925 Arg Cys Cys Glu Lys Thr Met Glu Val Met Arg Asn Ser Gln Glu 2930 2935 2940 Thr Leu Leu Thr Ile Val Glu Val Leu Leu Tyr Asp Pro Leu Phe 2945 2950 2955 Asp Trp Thr Met Asn Pro Leu Lys Ala Leu Tyr Leu Gln Gln Arg 2960 2965 2970 Pro Glu Asp Glu Thr Glu Leu His Pro Thr Leu Asn Ala Asp Asp 2975 2980 2985 Gln Glu Cys Lys Arg Asn Leu Ser Asp Ile Asp Gln Ser Phe Asp 2990 2995 3000 Lys Val Ala Glu Arg Val Leu Met Arg Leu Gln Glu Lys Leu Lys 3005 3010 3015 Gly Val Glu Glu Gly Thr Val Leu Ser Val Gly Gly Gln Val Asn 3020 3025 3030 Leu Leu Ile Gln Gln Ala Ile Asp Pro Lys Asn Leu Ser Arg Leu 3035 3040 3045 Phe Pro Gly Trp Lys Ala Trp Val 3050 3055 2 7 PRT Artificial Sequence Substrate recognition sequence. 2 Xaa Xaa Xaa Ser Gln Xaa Xaa 1 5 3 6 PRT Artificial Sequence Partial peptide sequence. 3 Xaa Glu Xaa Ser Xaa Lys 1 5 4 19 PRT Homo sapiens 4 Ser Leu Ala Phe Glu Glu Gly Ser Gln Ser Thr Thr Ile Ser Ser Leu 1 5 10 15 Ser Glu Lys 5 15 PRT Artificial Sequence Synthetic peptide. 5 Ser Leu Ala Phe Glu Glu Gly Ser Gln Ser Thr Thr Ile Ser Ser 1 5 10 15 6 16 PRT Artificial Sequence Synthetic peptide. 6 Ser Leu Ala Phe Glu Glu Gly Ser Pro Gln Ser Thr Thr Ile Ser Ser 1 5 10 15 

What is claimed is:
 1. A method of identifying the activation state of Ataxia-Telangiectasia Mutated kinase in a cell comprising determining the phosphorylation state of a serine corresponding to residue 1981 of Ataxia-Telangiectasia Mutated kinase of SEQ ID NO: 1 in a cell wherein the phosphorylation state of said serine is indicative of the activation state of Ataxia-Telangiectasia Mutated kinase.
 2. The method of claim 1 wherein, the cell is from a biological sample.
 3. The method of claim 1, wherein the phosphorylation state of the serine is determined using an antibody which specifically recognizes the phosphorylation state of said serine.
 4. The method of claim 3, wherein the antibody is detected using an immunoassay comprising enzyme-linked immunosorbent, immunodiffusion, chemiluminescent, immunofluorescent, immunohistochemical, radioimmunoassay, agglutination, complement fixation, immunoelectrophoresis, western blots, mass spectrometry, antibody array, or immunoprecipitation assays.
 5. An antibody specific for Ataxia-Telangiectasia Mutated kinase wherein the antibody specifically recognizes the phosphorylation state of a serine corresponding to residue 1981 of Ataxia-Telangiectasia Mutated kinase of SEQ ID NO:
 1. 6. The antibody of claim 5, wherein the antibody specifically recognizes a phosphorylated serine corresponding to residue 1981 of Ataxia-Telangiectasia Mutated kinase of SEQ ID NO:
 1. 7. The antibody of claim 6, wherein the antibody is a polyclonal antibody or a fragment thereof.
 8. The antibody of claim 6, wherein the antibody is a monoclonal antibody or a fragment thereof.
 9. The antibody of claim 5, wherein the antibody specifically recognizes an unphosphorylated serine corresponding to residue 1981 of Ataxia-Telangiectasia Mutated kinase of SEQ ID NO:
 1. 10. The antibody of claim 9, wherein the antibody is a polyclonal antibody or a fragment thereof.
 11. The antibody of claim 9, wherein the antibody is a monoclonal antibody or a fragment thereof.
 12. A method of detecting DNA damage in a sample comprising identifying the activation state of Ataxia-Telangiectasia Mutated kinase in a sample according to the method of claim 1, wherein the presence of active Ataxia-Telangiectasia Mutated kinase is indicative of DNA damage.
 13. The method of claim 12, wherein the sample comprises a biopsy sample, tissue, cell or fluid.
 14. The method of claim 13, wherein the sample is obtained from a subject exposed to radiation therapy or chemotherapy.
 15. A method of detecting a DNA damaging agent in a sample comprising contacting a cell containing Ataxia-Telangiectasia Mutated kinase with a sample suspected of having a DNA damaging agent and identifying the activation state of said kinase in the cell according to the method of claim
 1. 16. The method of claim 15, wherein the sample is a biological or environmental sample.
 17. A kit for the detecting a DNA damaging agent in a sample comprising an antibody of claim
 5. 18. The kit of claim 17 further comprising Ataxia-Telangiectasia Mutated kinase protein.
 19. A method of producing soluble Ataxia-Telangiectasia Mutated kinase or a fragment thereof comprising contacting a first polypeptide which contains the Ataxia-Telangiectasia Mutated kinase domain with a second polypeptide which contains a serine residue 1981 of Ataxia-Telangiectasia Mutated kinase of SEQ ID NO: 1 so that soluble Ataxia-Telangiectasia Mutated kinase is produced.
 20. The method of claim 19, wherein the first and second polypeptide are produced separately.
 21. The method of claim 19, wherein the first and second polypeptide are produced as a single polypeptide.
 22. The method of claim 19, wherein the first and second polypeptide are produced in the same cell.
 23. A method of identifying an agent that modulates the activation state of Ataxia-Telangiectasia Mutated kinase comprising contacting a cell containing Ataxia-Telangiectasia Mutated kinase with an agent and identifying the activation state of said kinase according to the method of claim
 1. 24. The method of claim 23, wherein the agent agonizes the activation of Ataxia-Telangiectasia Mutated kinase.
 25. The method of claim 23 further comprising contacting said cell with a DNA damaging agent.
 26. The method of claim 25, wherein the agent antagonizes the activation of Ataxia-Telangiectasia Mutated kinase.
 27. The method of claim 25, wherein the agent enhances the activation of ATM kinase.
 28. A method of identifying an agent that inhibits Ataxia-Telangiectasia Mutated kinase activity comprising contacting soluble Ataxia-Telangiectasia Mutated kinase protein with an agent and a phosphate donor and determining the phosphorylation state of a serine of said soluble Ataxia-Telangiectasia Mutated kinase corresponding to residue 1981 of Ataxia-Telangiectasia Mutated kinase of SEQ ID NO: 1 wherein the phosphorylation state of said serine is indicative of said kinase activity. 